CN1842515A - Cathepsin inhibitors - Google Patents

Cathepsin inhibitors Download PDF

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CN1842515A
CN1842515A CNA2004800245200A CN200480024520A CN1842515A CN 1842515 A CN1842515 A CN 1842515A CN A2004800245200 A CNA2004800245200 A CN A2004800245200A CN 200480024520 A CN200480024520 A CN 200480024520A CN 1842515 A CN1842515 A CN 1842515A
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C·拜利
C·布拉克
D·J·麦凯
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Merck Canada Inc
Merck Frosst Canada and Co
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Merck Frosst Canada Ltd
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Abstract

This invention relates to a novel class of compounds, represented by the formula (I) below, wherein the meanings of R1, R2, R3 and R4 are indicated therein, which are cysteine protease inhibitors, including but not limited to, inhibitors of cathepsins K, L, S and B. These compounds are useful for treating diseases in which inhibition of bone resorption is indicated, such as osteoporosis, osteoarthritis and rheumatoid arthritis.

Description

Cathepsin inhibitors
Technical field
The present invention relates in general to the inhibitor of protein active, and is particularly related to the inhibitor of kethepsin.
Background technology
Many kethepsins belong to the papoid superfamily of L-Cysteine HCL Anhydrous.These proteolytic enzyme play a role in degraded of the normal physiologic of reticular tissue and pathology degraded.Kethepsin is in intracellular protein degraded and renewal and play a part very important in reproducing.These kethepsins are present in the multiple tissue natively.Up to now, many kethepsins have obtained from many sources identifying and order-checking that for example, cathepsin B, F, H, L, K, S, W and Z have obtained the clone.In addition, disclose the sequence of cathepsin K among the PCT application WO 96/13523 of the KhepriPharmaceuticals company that publishes on May 9th, 1996, the full text of this application draws at this and is reference.There is complicated relation in cathepsin L with normal antalzyme protein matter hydrolysis and some morbid state (including but not limited to that melanoma shifts).There are complicated relation in cathepsin S and Alzheimer and some autoimmunity disorder (include but not limited to teenager show effect diabetes, multiple sclerosis, chronic pemphigus, hyperthyroidism, myasthenia gravis, systemic lupus erythematosus, rheumatoid arthritis and struma lymphomatosa), allergic disorder (including but not limited to asthma) and exogenous immune response (include but not limited to organ-graft refection or tissue transplantation repel).The cathepsin B's level that find to raise in tumour and the redistribution of enzyme show that this enzyme plays a role at tumor invasion with in shifting.In addition, there is complicated relation in unusual cathepsin B's activity with certain class morbid state (as rheumatoid arthritis, osteoarthritis, Pneumocystis carinii, acute pancreatitis, airway inflammatory disease and B﹠J illness).
Known cystatin such as E-64 (anti--epoxy succinic acyl-L-leucyl amine-(4-guanidine radicals) butane) can effectively suppress bone resorption.Consult Delaisse, people such as J.M., 1987, Bone, 8:305-313, it draws at this in full and is reference.Recently, cathepsin K has been carried out the clone and found that it is expressed in the osteoclast specifically.Consult Tezuka, people such as K., 1994, J Biol Chem, 269:1106-1109; People such as Shi G.P., 1995, FEBS Lett, 357:129-134; Bromme, D. and Okamoto, K., 1995, BiolChem Hoppe Seyler, 376:379-384; People such as Bromme D., 1996, J BiolChem, 271:2126-2132; Drake, people such as F.H., 1996, J Biol Chem, 271:12511-12516, it draws at this in full and is reference.Simultaneously with this clone be that it is relevant with sudden change in being present in this cathepsin K gene to have described autosomal recessive illness and pycnodysostosis (it is characterized in that the osteosclerosis phenotype that bone resorption dwindles).Up to now, known all sudden changes of identifying in the cathepsin K gene can cause the protein of inactivation.Consult Gelb, people such as B.D., 1996, Science, 273:1236-1238; Johnson, people such as M.R., 1996, Genome Res, 6:105O-1055, it draws at this in full and is reference.Therefore, as if the bone resorption with the osteoclast mediation is relevant for cathepsin K.
Cathepsin K is synthesized the 37kDa prozymogen, and this prozymogen is limited to the N,O-Diacetylmuramidase layer, and it has been generally acknowledged that it can be become sophisticated 27kDa enzyme by auto-activation under low pH value.Consult McQueney, people such as M.S., 1997, J Biol Chem, 272:13955-13960; Littlewood-Evans, people such as A., 1997, Bone, 20:81-86, it draws at this in full and is reference.Cathepsin K is closely related with the cathepsin S that 56% sequence identity is arranged on amino acid levels most.The S of cathepsin K 2P 2Substratspezifitaet is similar to cathepsin S, and is preferential on P1 and P2 position respectively for positively charged residue (for example arginine) and hydrophobic residue (for example phenylalanine or leucine).Consult Bromme, people such as D., 1996, J Biol Chem, 271:2126-2132; Bossard, people such as M.J., 1996, J Biol Chem, 271:12517-12524, it draws at this in full and is reference.Cathepsin K is having activity in the pH scope widely, has significant activity 4～8 times in the pH value, makes it have excellent catalytic activity thus in the absorbing cavity of pH value for about 4～5 osteoclast.
Human I type collagen, the main collagen in the bone is the good substrate of cathepsin K.Consult Kafienah, people such as W., 1988, Biochem J, 331:727-732, it draws at this in full and is reference.To the external bone resorption that cathepsin K has used the in vitro tests of antisense oligonucleotide to show to reduce, this may be because the minimizing of cathepsin K mRNA translation causes.Consult Inui, people such as T., 1997, J Biol Chem, 272:8109-8112, it draws at this in full and is reference.The crystalline structure of cathepsin K is resolved.Consult McGrath, people such as M.E., 1997, Nat Struct Biol, 4:105-109; Zhao, people such as B., 1997, Nat Struct Biol, 4:109-11, it draws at this in full and is reference.Equally, developed inhibitor based on the optionally cathepsin K of peptide.Consult Bromme, people such as D., 1996, Biochem J, 315:85-89; Thompson, people such as S.K., 1997, Proc Natl Acad Sci USA, 94:14249-14254, it draws at this in full and is reference.Therefore, cathepsin K inhibitor can reduce bone resorption.These inhibitor can be used for treating the illness that relates to bone resorption, for example osteoporosis.
Summary of the invention
The invention provides the compound that in the Mammals that needs treat and/or prevent, can treat and/or prevent kethepsin dependent status or disease attitude.The invention provides following general formula compound:
Figure A20048002452000071
R wherein 4Be non-hydrogen substituting group with electrophilic character, itself and R 1, R 2And R 2Be enough to together the pKa of nitrogen in the water medium is reduced to less than 6,
R wherein 1Be the S that is incorporated into the tissue protein zymophore 1The substituting group of sublocus and
R wherein 2Be the S that is incorporated into the tissue protein zymophore 2The substituting group of sublocus and
R wherein 3Be the S that is incorporated into the tissue protein zymophore 3The substituting group of sublocus.
More specifically, the invention provides following general formula compound:
Figure A20048002452000072
R wherein 1Be the S that is incorporated into the tissue protein zymophore 1The substituting group of sublocus and R wherein 2Be the S that is incorporated into the tissue protein zymophore 2The substituting group of sublocus and R wherein 3Be the S that is incorporated into the tissue protein zymophore 3The substituting group of sublocus.
In order to describe the reactive site of proteinase inhibitor, special nomenclature has been proposed.Consult U.S. Patent number 6,333,402, be hereby incorporated by.Residue from the amino side-chain of the key of the easy disconnection of substrate begins, and leaves from this key, and residue is named as P 1, P 2And P 3Deng.To follow the residue after scissile key to be called P 1', P 2' and P 3' etc.Have been found that the main chain with unusual protein inhibitor of different integral structure is at P 3And P 3' between the zone very similar, simultaneously for P 2, P 1And P 1' be especially highly similar.It is conventionally believed that each protease activity position has sublocus S 1And S 2Deng and sublocus S 1' and S 2' etc., S 1And S 2The residue P that holds substrate or inhibitor 1And P 2Deng side-chain radical, S 1' and S 2' hold the P of substrate or inhibitor 1And P 2Deng side-chain radical.Interaction between S sublocus and the P side-chain radical produces proteolytic enzyme for the specificity of substrate and the inhibitor specificity for proteolytic enzyme.This nomenclature is usually directed to the Nonpeptide inhibitors of proteolytic enzyme, wherein with proteolytic enzyme sublocus S 1And S 2P will be called as etc. interactional non-peptide zone respectively 1And P 2Deng.
P in the dipeptides cathepsin inhibitors 2-P 3Amido linkage can be replaced by substituted ethamine residue.R 1, R 2, R 2And R 4The electrophilic performance set get up to make P 4Amine becomes non-alkalescence under the physiological pH value.Thus, this fragment can form important neutral hydrogen bond with conservative kethepsin glycine in the whole papoid of the kethepsin family.
Acid amides and aniline can both form hydrogen bond with carbonyl, but have metabolic tendency.Ethamine itself be alkalescence and in biosystem by protonated, produce one and can reduce and target enzyme bonded electric charge.Has abundant electrophilic R 4When (perhaps more specifically being trifluoro ethamine), amine is not formed stronger hydrogen bond by the acceptor oxygen in protonated and amine and the tissue protein zymophore.In kethepsin, important acceptor oxygen is positioned at the S of reactive site 2And S 3Between the sublocus; Acceptor oxygen is Gly66 in cathepsin K.This residue is the conserved residues among proteinic this whole papoid family, and comprises cathepsin B, F, H, K, L, L 2, O, S, W and Z, falcipain, falcipain-1 and falcipain-2.Therefore, use non-alkaline ethamine connexon to connect and cathepsin S 2And S 3Sublocus bonded inhibitor fragment can produce than using the more efficiently restraining effect of corresponding amides.
Detailed Description Of The Invention
The present invention relates to have the composition of the material of following chemical formula:
Figure A20048002452000081
Be no more than 70 non-hydrogen atoms that are selected from C, O, N, S, P, F, Cl, Br or I independently of one another with having;
R wherein 4Be non-hydrogen electron-withdrawing substituent, thereby make itself and R 1, R 2And R 2Make the alkalescence of nitrogen be reduced to pKa together less than 6;
Wherein composition molecule and tissue protein enzyme interacting make CH-NH zone and kethepsin in the chemical formula at S 2And S 3Between advantageously interact R 1S with the tissue protein zymophore 1But not S 3Advantageously interact R 2S with the tissue protein zymophore 2But not S 3Advantageously interact, and R 2S with the tissue protein zymophore 3But not S 2Perhaps S 1Advantageously interact.
In one group of technical scheme of the present invention, kethepsin is selected from cathepsin B, F, H, K, L, L 2, O, S, W and Z.In a subgroup of the present invention, kethepsin is selected from cathepsin K, L, S and B.In another subgroup of the present invention, kethepsin is a cathepsin L.In another subgroup of the present invention, kethepsin is a cathepsin S.In another subgroup of the present invention, kethepsin is a cathepsin B.In another subgroup of the present invention, kethepsin is kethepsin F.
In one group of technical scheme of the present invention, R 4Not respectively with the sublocus S of tissue protein zymophore 2, S 3And S 1Advantageously interact.Of the present invention another kind of in, R 4Be selected from-CF 3,-CH 2F ,-CF 2R 5With-CHFR 5, R wherein 5Be the C that is randomly replaced by 1～4 substituting group 1-6Alkyl, aryl or heteroaryl, these substituting groups are selected from halogen, C 1-3Alkyl, C 1-3Alkoxyl group, hydroxyl, hydroxyalkyl, ketone, cyano group, heterocyclic radical, C 3-8Cycloalkyl, SO mC 1-3Alkyl, NH 2, NO 2Perhaps O (C=O) C 1-3Alkyl, wherein m is 0～2 integer.
In one group of technical scheme of the present invention, R 2Have at least one carbon atom that satisfies following three criterion distance simultaneously or sulphur atom, this criterion distance is: it is in cathepsin C α 267  scopes in, in cathepsin C α 688.5  scopes in and in cathepsin C α 1347  scopes in.Of the present invention another kind of in, R 2Comprise apolar regions.Of the present invention another kind of in, R 2Comprise lipophilic zone.
In one group of technical scheme of the present invention, R 1Comprise stably sublocus S with the tissue protein zymophore 1The zone that cooperates has at least one in cathepsin C α 255  scopes in carbon atom.Of the present invention another kind of in, R 1Right and wrong are immunogenic.
In one group of technical scheme of the present invention, R 2Have at least one carbon atom that satisfies following two criterion distance simultaneously or sulphur atom, this criterion distance is: it is in cathepsin C α 667  scopes in and in cathepsin C α 607  scopes in.Of the present invention another kind of in, R 2Comprise apolar regions.Of the present invention another kind of in, R 2Comprise lipophilic zone.
In one group of technical scheme of the present invention, the pKa of nitrogen is less than 6 and form a hydrogen bond with the kethepsin amidocarbonylation of kethepsin glycine 66.
In one group of technical scheme of the present invention, compound has less than 1000 daltonian molecular weight.In one group of technical scheme of the present invention, the halfcystine 25 of compound and kethepsin forms covalent linkage.In one group of technical scheme of the present invention, compound combines with the reactive site of kethepsin, IC in the enzyme assay of purifying 50Less than 10 micromoles.
In one group of technical scheme of the present invention, at the pKa of secondary amine nitrogen in water medium shown in the claim 1 less than 5.
The term of Shi Yonging " lipophilic " is meant a kind of compound as separate entity in this article, and is dissolved in water than more being soluble in non-polar solvent (for example hexanaphthene).Term " oleophilic group ", it is connected on the molecule in context, is meant a zone of the molecule with high hydrocarbon content, thereby makes group produce high affinity to non-polar solvent or lipid.Oleophilic group can be for example to be less than the alkyl chain or the cycloalkyl chain (being preferably positive alkyl) of 30 carbon atoms.Further, oleophilic group comprises the alkyl chain of and the non-aromatic portion fragrant with synthetic that is connected in natural formation, for example lipid acid, ester and alcohol and other lipid molecule.Other example of lipophilic molecules be cage structure (for example diamantane and buckminsterfullerence) and aromatic hydrocarbon (for example Ben, perylene, phenanthrene, anthracene, naphthalene, pyrene, With four acenes).Term " oleophilic group " is particularly including the carbon atom and the hydrogen atom that is connected of alkyl chain, cycloalkyl chain, aromatic ring or hetero-aromatic ring as used herein.Term " oleophilic group " also comprises bivalent sulfur atom as used herein.
Term " homologous basically " when using together with aminoacid sequence, is meant basic identical or similar sequence on sequence, and this causes the conformation homology and causes similar biological activity thus.The implicit common sequence of not planning this term originates from.Generally " homologous basically " sequence is on order, in any known relating on the active zone of expectation, has at least 50% sequence identical at least, and more preferably at least 80% is identical.Most preferably except end group, five residue differences are arranged at the most.Preferably be " conservative distortion " form at least in aforementioned region in the different difference on the sequence.
" conservative distortion " is defined as (a) amino acid whose conservative substitution as hereinafter defining; (b) endways, the region intermediate border, in ring or the part of other high relatively reactivity (for example by X-ray diffraction analysis or NMR can't know parsing shown in part), amino acid whose single or multiple insertion or cancellation.Preferably except endways, about at the most five amino acid is inserted into or cancellation at specific site, and deformation is to contain the active and external region combining site wanted of overstating.
Conservative substitution is defined herein as in following five groups one group and exchanges, and five groups are: the nonpolar or slight polar residue of aliphatics that (1) is little: L-Ala (Ala), Serine (Ser) and Threonine (Thr) (proline(Pro) (Pro), glycine (Gly)); (2) residue of polarity zone negative charge and their acid amides: aspartic acid (Asp), l-asparagine (Asn), L-glutamic acid (Glu) and glutamine (Gln); (3) the positively charged residue of polar: Histidine (His), arginine (Arg) and Methionin (Lys); (4) big aliphatics non-polar residue: methionine(Met) (Met), leucine (Leu), Isoleucine (Ile) and Xie Ansuan (Val) (halfcystine (Cys)); (5) big aromatic residues: phenylalanine (Phe), tyrosine (Tyr) and tryptophane (Trp).It is because they have special conformation effect that residue Pro, Gly and Cys have drawn together with parenthesis.Cys participates in the formation of disulfide linkage.Glycine is given chain with flexible.Proline(Pro) is given chain with rigidity and destruction alpha-helix.These residues may be basic residues in some zone of polypeptide, are interchangeable in other place still.Half conservative substitution is defined in the exchange between two groups of above-mentioned (1)～(5) groups, and above-mentioned five groups can be defined as preeminent (a), comprise above-mentioned group (1), (2) and (3), perhaps are defined as preeminent (b), comprise above-mentioned group (4) and (5).
Term " kethepsin " is meant the enzyme that belongs to papoid family as used herein, promptly wherein the activity form of enzyme with the mode that is similar to papoid fold (be Conserved Domain smart00645.7 in NCBIConserved Domain database, http: // Www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi? uid=15045).Only relate to kethepsin in the mankind, mouse, rabbit, primate, rat and the little sickle plasmodium at this.Human tissue proteolytic enzyme is particularly including cathepsin B, F, H, K, L, L 2, O, S, W and Z; The enzyme in mouse, rabbit, primate and the rat also, these enzymes are compared with the most similar formerly described human tissue proteolytic enzyme to demonstrate and are surpassed 80% sequence identity (the relatively activity form of enzyme).Also falcipain, falcipain-1 and the falcipain-2 in the little sickle plasmodium with the most similar to these enzymes, demonstrates any other the little sickle plasmodium enzyme that surpasses 80% sequence identity, equally relatively the activity form of enzyme.
Specified amino acid residues in the kethepsin (for example glycine 66) or C α (C α for example 66) be meant combination by the residue numbering that is used for the basic sequence comparison that cathepsin K activity form and Fig. 1 provide.This sequence alignment is used for the relevant portion as the protein active form of the human tissue proteolytic enzyme of figure mark and falcipains.In the accompanying drawings, provided some Key residues numberings that relate to herein, layout becomes the form perpendicular to the sequence of above-mentioned comparison.Other residue numbering that relates to can be by calculating along immediate numbering residue or obtaining by direct reference tissue Proteinase K sequence.Numbering is that benchmark obtains with the KSWISSPROT master index registration #P43235 of human tissue proteolytic enzyme, first residue in its medium chain, and activity of proteins form (residue is numbered 115 in #P43235) resets to residue number 1 at this.Be used to of the reference of organization of unity Proteinase K residue at this sequence alignment for another human kethepsin and falcipains.The basic sequence comparison that the reference of the residue in mouse, rabbit, primate and the rat tissue's proteolytic enzyme is used the standard of default parameter imply, extremely in the sequence of the following specifically qualification of homologous:
22
56
**
Figure A20048002452000121
6 667
0 680
* ***
Cathepsin K: YPYVG---------QEESCMYNPT-------------------GKAAKCR
Cathepsin B: YESHVGCRPYSIPPCEHHVNGSRPPCTGEGDTPKCSKICEPGYSPTYKQD
Kethepsin F:YSYQG---------HMQSCNFSAE-------------------KAKVYlN
Cathepsin H: YPYQG---------KDGYCKFQPG-------------------KAIGFVK
Cathepsin L: YPYEA---------TEESCKYNPK-------------------YSVANDT
Kethepsin O:YPFKA---------QNGLCHYFSG-------------------SHSGFSI
Cathepsin S: YPYKA---------MDQKCQYDSK-------------------YRAATCS
Kethepsin W:YPFQGK--------VRAHRCHPKKY------------------QKVAWIQ
Kethepsin Z:NNYQAKDQECDKFNQCGTCNEFKE-------------------CHAIRNY
The 2:YPYVA--------VDEICKYRPE-------------------NSVANDT of cathepsin L
Falcipain：YKYKAK--------DDMFCLNYRC-------------------KRKVSLS
Falcipain2：YPYVSN--------LPETCNLKRC-------------------NERYTIK
Falcipain3：YPYVSD--------APNLCNIDRC-------------------TEKYGIK
1
3
4
*
Cathepsin K: CNS-----DNLNHAVLAVGYGIQKG-------------------------
Cathepsin B: GEMMG------GHAIRILGWGVENG-------------------------
Kethepsin F:CSP-----WLIDHAVLLVGYGNRSD-------------------------
Cathepsin H: CHKTP---DKVNHAVLAVGYGEKNG-------------------------
Cathepsin L: CSS-----EDMDHGVLVVGYGFESTE---------------------SDN
Kethepsin Q:GE--------ANHAVLITGFDKTGS-------------------------
Cathepsin S: CT------QNVNHGVLVVGYGDLNG-------------------------
Kethepsin W:CDP-----QLVDHSVLLVGFGSVKSEEGIWAETVSS-----QSQPQPPHP
Kethepsin Z:DTT------YINHVVSVAGWGISDG-------------------------
The 2:CSS-----KNLDHGVLVVGYGFEGAN--------------------SNN of cathepsin L
Falcipain：SEE-------LNHSVLLVGYGQVEKTKLNYNNKIQTYNTKENSNQPDDNI
Falcipain2：GA------A-PNHAVILVGYGMKDIYNEDTG---------------RMEK
Falcipain3：GD------E-LNHAVMLVGFGMKEIVNPLTK---------------KGEK
2
0
9
*
Cathepsin K: NKHWIIKNSWGENWGNKGYILMARNKN-------NACGIANLASFPKM--
Cathepsin B: TPYWLVANSWNTDWGDNGFFKILRGQD--------HCGIESEVVAGIPRT
Kethepsin F:VPFWAIKNSWGTDWGEKGYYYLHRGSG--------ACGVNTMASSAVVD-
Cathepsin H: IPYWIVKNSWGPQWGMNGYFLIERGK--------NMCGLAACASYPIPLV
Cathepsin L: NKYWLVKNSWGEEWGMGGYVKMAKDRR-------NHCGIASAASYPTV--
Kethepsin O:TPYWIVRNSWGSSWGVDGYAHVKMGSN--------VCGIADSVSSIFV--
Cathepsin S: KEYWLVKNSWGHNFGEEGYIRMARNKG-------NHCGLASFPSYPEI--
Kethepsin W:TPYWILKNSWGAQWGEKGYFRLHRGSN--------TCGiTKFPLTARVQK
Kethepsin Z:TEYWIVRNSWGEPWGERGWLRIVTSTYKDGKGARYNLAIEEHCTFGDPIV
The 2:SKYWLVKNSWGPEWGSNGYVKIAKDKN-------NHCGIATAASYPNV--of cathepsin L
Falcipain：IYYWIIKNSWSKKWGENGFMRLSRNKNGD----NVFCGIGEEVFYPIL--
Falcipain2：FYYYIIKNSWGSDWGEGGYINLETDENGY----KKTCSIGTEAYVPLLE-
Falcipain3：HYYYIIKNSWGQQWGERGFINIETDESGL----MRKCGLGTDAFIPLIE-
Cathepsin K:------------(SEQ ID NO:1)
Cathepsin B: D-----------(SEQ ID NO:2)
Kethepsin F:------------(SEQ ID NO:3)
Cathepsin H:------------(SEQ ID NO:4)
Cathepsin L:------------(SEQ ID NO:5)
Kethepsin O:------------(SEQ ID NO:6)
Cathepsin S:------------(SEQ ID NO:7)
Kethepsin W:PDMKPRVSCPP-(SEQ ID NO:8)
Kethepsin Z:------------(SEQ ID NO:9)
The 2:------------of cathepsin L (SEQ ID NO:10)
Falcipain：------------(SEQ?ID?NO：11)
Falcipain2：------------(SEQ?ID?NO：12)
Falcipain3：------------(SEQ?ID?NO：13)
Fig. 1
Definition: term " advantageously ", " advantageously interact " as being used for term in this article, be meant and make the advantageous results that molecular model calculates, wherein the computer model of ligand molecular is inserted the reactive site of kethepsin computer model, produce part: the kethepsin title complex, and use the standard molecule mechanics field of force, MMFF94s part for example: the energy minimization (T.A.Halgren of kethepsin title complex, Journal of Computational Chemistry (1999), 20, the 720-729 page or leaf).Part enters the initial layout within the reactive site, be also referred to as " butt joint " and carry out like this so that optimum make part: enzyme interacts between the sublocus of part substituting group and enzyme described herein.For the kethepsin of being studied (removing the nonprotein atom), if its x-ray crystal structure is obtainable, the computer model of reactive site is based on x-ray crystal structure (for example, for cathepsin K, Protein Databank inlet is 1MEM) so; If perhaps x-ray crystal structure is unavailable, just can use with the homology structural texture of the most similar kethepsin as benchmark, for this most similar kethepsin, its x-ray crystal structure is obtainable; Perhaps only directly using its x-ray structure is the most similar obtainable kethepsin.When energy minimization begins, also do not regulate the protein atom to be adapted to part, still during energy minimization, allow to have the whole protein side chain of at least one atom within part 6  and move along with the carrying out of energy minimization.During energy minimization, do not allow the backbone atoms of enzyme to produce mobile.Can make the continuous electric dielectric solvent of water, but can use the default parameter and the running condition of the molecular mechanics energy minimization of standard in addition, it to the software program macromodel of SchrodingerInc in the default parameter that adopts similar with running condition." advantageous results that Modelling is calculated " is meant after energy minimization is finished there is not bad steric interaction between part and reactive site, and exists the hydrogen bond of stable energy and oleophylic to interact between part and reactive site.Form covalent linkage if believe the reactive site sulphur of part and halfcystine-25, so energy minimization can comprise submit to calculate at the simulation part: the covalent linkage in the kethepsin title complex.As the part that requires herein and the interaction between the kethepsin based on part: the geometry of kethepsin title complex, this geometry is produced by above-mentioned energy minimization.
In one embodiment of the invention, part substituting group and S 2Between favourable interaction need substituent at least one carbon atom or bivalent sulfur atom to satisfy following three distance conditions simultaneously: it is at the α of cathepsin C 267  scopes in, at the α of cathepsin C 688.5  scopes in and at the α of cathepsin C 1347  scopes in.In another embodiment of the present invention, part substituting group and S 2Between favourable interaction need in the 5.5  scopes of atom of at least one atom two residues in residue 67,68 and 134 of substituting group oleophilic group.Further only relate to cathepsin B in the embodiment in the present invention, part substituting group and S 2Between favourable interaction need between the L-glutamic acid 209 of substituting group and cathepsin B, have hydrogen bond.
In one embodiment of the invention, part substituting group and S 3Between favourable interaction need substituent at least one carbon atom or bivalent sulfur atom to satisfy following two distances simultaneously: it is at the α of cathepsin C 665.5  scopes within, and it is within 7  scopes of kethepsin cage.In another embodiment of the present invention, part substituting group and S 3Between favourable interaction need at least one atom of substituting group oleophilic group at C α 66In 5.5  scopes of residue 60 or 61 atoms.In another embodiment of the present invention, part substituting group and S 3Between favourable interaction need at least one atom of substituting group oleophilic group in 5.5  scopes of residue 60 or 61.
In one embodiment of the invention, part substituting group and S 1Between favourable interaction need substituent at least one carbon atom at C α 255  scopes in.In another embodiment of the present invention, part substituting group and S 1Between favourable interaction, need between the reactive site hcy thiolactone of residue 25 and part electrophilic carbon, a covalent linkage be arranged, be preferably carbonyl carbon or nitrile carbon.
In one embodiment of the invention, CH-NH zone and S in the chemical formula of part 2With S 3Between kethepsin between favourable interaction, the N that needs this chemical formula is in 4  scopes of the peptide oxygen of Gly66, and the C of this chemical formula (only relates to directly and R 4The C that links to each other) at C α 665.5  scopes in.In another embodiment of the present invention, CH-NH zone and S in the part chemical formula 2With S 3Between kethepsin between favourable interaction, need allow the NH hydrogen bond of this chemical formula be attached on the acid amides 0 of residue 66.
Unless otherwise mentioned, " alkyl " used herein refers to and comprises the side chain with 1～10 carbon atom and the radical of saturated aliphatic hydrocarbyl group of straight chain.For example, C 1～C 10, as at " C 1～C 10Alkyl " in, be defined as and be included in the group that has 1,2,3,4,5,6,7,8,9 or 10 carbon atom in straight chain, side chain or the circular permutation.For example, " C 1～C 10Alkyl " particularly including methyl, ethyl, propyl group, butyl, amyl group, hexyl, heptyl, octyl group, nonyl and decyl etc.
Unless otherwise mentioned, " alkoxyl group " represents alkyl as defined above, and wherein said alkyl connects by oxo bridge.
Unless otherwise mentioned, term " cycloalkyl " or " carbocyclic ring " are meant that the total number of carbon atoms is the cyclic alkane ring (being cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, suberyl or ring octyl group) of any number in 3～8 or this scope.
" aryl " used herein is meant any stable monocycle or the two ring carbocyclic rings that are up to 12 atoms in each ring, wherein at least one ring is an aromatic nucleus.The example of above-mentioned aryl unit comprises phenyl, naphthyl, tetralyl, 2,3-indanyl, xenyl, phenanthryl, anthryl or acenaphthenyl.Be appreciated that at aryl substituent it is that bicyclic substituting group and a ring are in the situation of non-aromatic ring, connection is undertaken by aromatic ring.
Have the stable monocycle, two ring or three rings that are up to 10 atoms in each ring of term used herein " heteroaryl " expression, wherein at least one ring is aromatic nucleus and contains 1～4 heteroatoms that is selected from O, N and S.Heteroaryl groups in this range of definition includes but not limited to: benzimidazolyl-, benzofuryl, benzo furazan base, the benzopyrazoles base, the benzotriazole base, benzothienyl, the benzoxazol base, carbazyl, carbolinyl, the cinnolines base, furyl, indolinyl, indyl, indolizine base (indolazinyl), indazolyl, isobenzofuran-base, pseudoindolyl, isoquinolyl, isothiazolyl, different  azoles base, the naphtho-pyridyl, the  di azoly,  azoles base,  azoles quinoline, different  azoles quinoline, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, the pyridopyridine base, pyridyl, pyrimidyl, pyrryl, quinazolyl, quinolyl, quinoxalinyl, tetrazyl, the tetrazolo pyridyl, thiadiazolyl group, thiazolyl, thienyl, triazolyl, the dihydrobenzo imidazolyl, dihydro benzo furyl, the dihydrobenzo thienyl, dihydrobenzo  azoles base, indolinyl, the dihydroquinoline base, methylenedioxyphenyl, benzothiazolyl, benzothienyl, quinolyl, isoquinolyl,  azoles base and tetrahydric quinoline group.The heteroaryl substituting group is that two ring substituents and a ring are non-aromatic rings or do not comprise under the heteroatomic situation therein, should be appreciated that respectively to connect by aromatic ring or by the heteroatoms that comprises ring.If heteroaryl contains nitrogen-atoms, be to be understood that also to comprise its corresponding N-oxide compound in this definition.
Understand as those skilled in the art, " halo " used herein or " halogen " mean and comprise chlorine, fluorine, bromine and iodine.Term " ketone " is meant carbonyl (C=O).Term used herein " alkoxyl group " refers to by Sauerstoffatom and is connected to moieties on the remainder of molecule, and wherein alkyl as defined above.The example of alkoxyl group comprises methoxyl group and oxyethyl group etc.
Term " hydroxyl base alkyl " is meant the straight chain univalence hydrocarbyl of 1～6 carbon atom that is replaced by one or two hydroxyl or the side chain univalence hydrocarbyl of 3～6 carbon atoms, and condition is that they are not on same carbon atom if there are two hydroxyls.Representational example includes but not limited to methylol, 2-hydroxyethyl, 2-hydroxypropyl and 3-hydroxypropyl etc.
Unless otherwise mentioned, term used herein " heterocycle " or " heterocyclic radical " represent that containing 1～4 is selected from O, N, S, SO or SO 2Heteroatomic 5～10 yuan of non-aromatic rings, and comprise bicyclic groups.Therefore, " heterocyclic radical " includes but not limited to following group: piperazinyl, piperidyl, pyrrolidyl, morpholinyl, thio-morpholinyl, THP trtrahydropyranyl, dihydro piperidyl and tetrahydro-thienyl etc.If heterocycle contains nitrogen, be appreciated that in this definition also to comprise its corresponding N-oxide compound.
The present invention also comprises the N-oxide derivative and the shielded derivative of formula I compound.
For example, when formula I compound contains oxidable nitrogen-atoms, can nitrogen-atoms be changed into the N-oxide compound by method well known in the art.Also have when formula I compound contains hydroxyl for example, carboxyl, sulfydryl or any group that contains nitrogen-atoms, these groups can be protected with suitable protecting group.The comprehensive list of due care base can be at T.W.Greene, Protective Groups in Organic Synthesis, John Wiley ﹠amp; Sons, Inc. obtains in 1981, and its disclosed full content is hereby incorporated by.The shielded derivative of formula I compound can be by method preparation well known in the art.
Also comprise pharmaceutical composition within the scope of the present invention, pharmaceutical composition comprises aforesaid compound and pharmaceutically acceptable carrier.The present invention expects also and comprises a kind of pharmaceutical composition that it comprises pharmaceutically acceptable carrier and any clear and definite in this application disclosed compound.According to the instruction that is included in this, these and other aspect of the present invention will be conspicuous.
The The compounds of this invention pharmacy acceptable salt comprises the conventional non-toxic salt of the The compounds of this invention of inorganic or organic acid form.For example, conventional non-toxic salt comprises the salt that is obtained by mineral acid, hydrochloric acid for example, Hydrogen bromide, sulfuric acid, thionamic acid, the salt of phosphoric acid and nitric acid etc., and by the salt of organic acid preparation, for example acetic acid, propionic acid, succsinic acid, oxyacetic acid, stearic acid, lactic acid, oxysuccinic acid, tartrate, citric acid, xitix, pounce on acid (pamoic), toxilic acid, hydroxymaleic acid, phenylacetic acid, L-glutamic acid, M-nitro benzoic acid, Whitfield's ointment, sulfanilic acid, 2-acetoxyl group-M-nitro benzoic acid, fumaric acid, toluenesulphonic acids, methylsulfonic acid, ethylene disulfonic acid, oxalic acid, the salt of isethionic acid and trifluoroacetic acid etc.People such as Berg, ＂ Pharmaceutical Salts ＂, J.Pharm.Sci., 1977,66:1-19 has carried out describing more all sidedly to the preparation of above-mentioned pharmacy acceptable salt and other general pharmacy acceptable salt, is hereby incorporated by.The pharmacy acceptable salt of The compounds of this invention can utilize the conventional chemical method to be synthesized by the The compounds of this invention that contains alkalescence or acidic moiety.Usually, the salt of basic cpd or by ion exchange chromatography perhaps by in appropriate solvent or the combination of multiple solvent, makes free alkali and stoichiometric quantity or expects that with excessive formation the mineral acid or the organic acid reaction of salt prepare.Similarly, the salt of acidic cpd is by acidic cpd and suitable inorganic or organic bases reaction formation.
The compounds of this invention is a cathepsin inhibitors, and therefore has the purposes of treatment or prevention mammalian tissues proteolytic enzyme dependence disease or state, and Mammals is preferably human.
Especially, The compounds of this invention is a cathepsin K inhibitor, and the purposes that therefore has treatment or prevent mammiferous cathepsin K dependence disease or state, and Mammals is preferably human.
" kethepsin dependence disease or state " is meant and relies on the active pathologic state of one or more kethepsins." cathepsin K dependence disease or state " is meant and relies on the active pathologic state of one or more cathepsin Ks.Comprise osteoporosis with the active relevant disease of cathepsin K, the osteoporosis that the suprarenal gland glucocorticosteroid brings out, osteitis deformans, abnormal bone metabolism raises, periodontopathy, the tooth loss, fracture, rheumatoid arthritis, osteoarthritis, the Periprosthetic osteolysis, incomplete osteogenesis, obesity, atherosclerosis, chronic obstructive pulmonary disease, teenager's diabetes of showing effect, multiple sclerosis, chronic pemphigus, hyperthyroidism, myasthenia gravis, systemic lupus erythematosus, rheumatoid arthritis and struma lymphomatosa, asthma, exogenous immune response, parasitic infection, cancer, metastatic bone disease, malignant hypercalcemia or multiple myeloma.In these states of compounds for treating that require with the present invention, the therapeutic dose that needs will be according to disease specific and difference, and those skilled in the art can determine it at an easy rate.Though treatment and prevention can obtain expection in the scope of the invention, the treatment of these states is preferred purposes.
One embodiment of the invention be a kind of in the Mammals that needs the inhibition of histone enzymic activity method of inhibition of histone enzymic activity, comprise any as mentioned above compound or any pharmaceutical composition to Mammals drug treatment significant quantity.
One class of embodiment is a method, and wherein the tissue protein enzymic activity is the cathepsin K activity.
Another embodiment of the invention is a kind of method for the treatment of or preventing the kethepsin dependent status in the Mammals of needs treatment or prevention kethepsin dependent status, comprises any as mentioned above compound or any pharmaceutical composition to Mammals drug treatment significant quantity.
One class of embodiment is a method, and wherein the tissue protein enzymic activity is the cathepsin K activity.
Another embodiment of the present invention is a kind of method that suppresses the bone loss in the Mammals of needs inhibition bone loss, comprises any as mentioned above compound or any pharmaceutical composition to Mammals drug treatment significant quantity.Another embodiment of the present invention is a kind of method that reduces the bone loss in the Mammals of needs reduction bone loss, comprises any as mentioned above compound or any pharmaceutical composition to Mammals drug treatment significant quantity.The application of cathepsin K inhibitor in bone resorption suppresses is known in the literature.Consult people such as Stroup G.B., " Potent and selective inhibition of human cathepsin Kleadsto inhibition of bone resorption in vivo in a nonhumanprimate ", J.Bone Miner.Res., 16:1739-1746,2001; And Votta, B.J. wait the people, " Peptide aldehyde inhibitors of cathepsin Kinhibit boneresorption bone resorption both in vivo and in vitro ", J.BoneMiner.Res., 12:1396-1406,1997.
Another embodiment of the invention be a kind of in the Mammals of needs treatments or preventing osteoporosis disease the method for treatment or preventing osteoporosis disease, comprise any compound as mentioned above or any aforementioned pharmaceutical compositions to Mammals drug treatment significant quantity.The application of cathepsin K inhibitor in osteoporosis treatment or prevention is known in the literature.Consult people such as Saftig P., " Impaired osteoclast bone resorption leadsto osteoporosis in cathepsin K-deficient mice ", Proc.Natl.Acad.Sci., USA, 95:13453-13458,1998.
Another embodiment of the invention is a kind of method for the treatment of or preventing the rheumatic arthritis state in the Mammals of needs treatment or prevention rheumatic arthritis state, comprises any as mentioned above compound or any pharmaceutical composition to Mammals drug treatment significant quantity.In the literature, the progressive destruction of known joint surrounding bone is the unable major cause of joint dysfunction and joint among rheumatic arthritis (RA) patient.Consult Goldring SR, " Pathogenesis of bone erosions in rheumatoid arthritis ", Curr.Opin.Rheumatol., 14:406-10,2002.To analyzing, proved that the positive osteoclast of cathepsin K is the cell type of the mediation focus bone resorption relevant with the infringement of rheumatic synovia from joint tissue with patient RA.Consult Hou, people such as W-S, " Comparision of Cathepsin K and S expression within theRheumatoid and Osteoarthritic Synovium ", ArthritisRheumatism, 46:663-74,2002.In addition, general bone loss is the major cause that causes with serious RA pathologies associated.In having the patient of chronic RA, hall portion and spinal fracture frequency obtain increasing basically.Consult people such as Gould, " Osteoclastic activationis the principal mechanism leading to secondary osteoporosisin rheumatoid arthritis ", J.Rheumatol., 25:1282-9,1998.Cathepsin K inhibitor is bone resorption and the treatment of general bone loss or the application in the prevention under the joint, represents a kind of rational method that pharmacology is got involved in rheumatic arthritis increases the weight of.
Another embodiment of the invention is a kind of method for the treatment of or preventing osteoarthritis to carry out in the Mammals of needs treatment or prevention osteoarthritis, comprises any as mentioned above compound or any pharmaceutical composition to Mammals drug treatment significant quantity.In the literature, known bone sacroiliitis (OA) has clear and definite variation in the joint, comprises endochondral ossification/osteophytosis and subchondral bone sclerosis and sporangiocyst formation around articular cartilage surface erosion, the joint, consults Oettmeier, R ﹠amp; Abendroth, K, " Osteoarthritis and bone:osteologictypes of osteoarthritis of the hip ", Skeletal Radiol., 18:165-74,1989.Recently, having proposed subchondral bone sclerosis may impel OA to begin and carry out.When the load of joint response multiple pulsed, the subchondral bone of hardening can only weaken lessly and scatter the pressure by the joint, makes this pressure bigger in the mechanical stress of articular cartilage surface.This has also promoted cartilage wearing and tearing and fibrillation conversely.Consult Radin EL and RoseRM, " Role of subchondral bone in the initiation and progressionof cartilage damage ", Clin.Orthop., 213:34-40,1986.By suppress bone resorption under the too much joint with anti-absorption reagent (for example cathepsin K inhibitor), will cause the subchondral bone metabolism to be suppressed, may produce favorable influence to carrying out property OA thus.Except that above-mentioned hypothesis, recently, in similar cell of synovia inoblast, scavenger cell that picks up from patient's OA synovial membrane and joint cartilage sample and chondrocyte, determined the protein expression of cathepsin K.Consult Hou, people such as W-S, " Comparison of Cathepsin Kand S expression within the Rheumatoid and OsteoarthriticSynovium ", Arthritis Rheumatism, 46:663-74,2002; With people such as Dodd RA, " Expression of Cathepsin K messenger RNA in giant cellsand their precursors in human osteoarthritic synovialtissues ", Arthritis Rheumatism, 42:1588-93,1999; With people such as Konttinen, " Acidic cysteine endoproteinase cathepsin K in thedegeneration of the superficial articular hyaline cartilagein osteoarthritis ", Arthritis Rheumatism, 46:953-60,2002.Thus, these researchs recently relate to the effect of cathepsin K in the joint cartilage II Collagen Type VI that carries out with osteoarthritis destroys.Thus, the application of cathepsin K inhibitor in osteoarthritis treatment or prevention comprises two kinds of different mechanism as described in the present invention, a kind of mechanism is that to suppress subchondral bone metabolism that osteoclast drives and second kind of mechanism be directly to suppress to have patient's the synovial membrane of OA and the II Collagen Type VI sex change in the cartilage.
Another embodiment of the present invention is a kind ofly to treat method for cancer in the Mammals of needs treatments cancer, comprises any as mentioned above compound or any pharmaceutical composition to Mammals drug treatment significant quantity.The known tissue Proteinase K is expressed in human breast cancer in the literature.Consult people such as Littlewood-Evans AJ, " The osteoclast-associatedprotease cathepsin K is expressed in human breast carcinoma ", Cancer Res, 57 (23): 5386-90, on December 1st, 1997.
The purposes that example of the present invention is any compound as mentioned above in the preparation medicine, medicine are used for treating and/or preventing osteoporosis its Mammals of needs.The purposes that another specific examples of the present invention is any compound as mentioned above in the preparation medicine, medicine are used for the treatment of and/or prevent bone loss, bone resorption, fracture, metastatic bone disease and/or the illness relevant with the kethepsin function.
The compounds of this invention can be to Mammals, preferred human administration, individually dosed or preferred and pharmaceutically acceptable carrier or thinner Combined Preparation, in pharmaceutical composition, according to the standard drug practice, optional and known assistant agent Combined Preparation, assistant agent for example is an alum.Compound can be taken orally or parenteral admin, comprises intravenous, intramuscular, endoperitoneal, subcutaneous, rectum and partial route of administration.
Under the tablet situation that is used to orally use, normally used carrier comprises lactose and W-Gum, and adds lubricant, for example Magnesium Stearate usually.For the oral administration of capsule form, useful thinner comprises lactose and dried corn starch.In order to orally use according to treatment compound of the present invention, the administration in the following manner of the compound of selection for example with tablet or capsule form, perhaps forms the aqueous solution or suspension.For with tablet or capsule form oral administration, active constituents of medicine can be united use with oral, nontoxic, pharmaceutically acceptable inert support, and inert support for example is lactose, starch, sucrose, glucose, methylcellulose gum, Magnesium Stearate, secondary calcium phosphate, calcium sulfate, N.F,USP MANNITOL and Sorbitol Powder etc.; For with the liquid form oral administration, oral drug component can be united use with any oral, nontoxic, pharmaceutically acceptable inert support, and inert support is ethanol, G ﹠ W etc. for example.In addition, when expecting or needing, also can add suitable binder, lubricant, disintegrating agent and tinting material in the mixture.Suitable adhesive comprises starch, gelatin, natural carbohydrate (for example glucose or beta lactose), corn sweetener, natural gum and synthetic gum (for example gum arabic, tragakanta or sodium alginate), carboxymethyl cellulose, polyoxyethylene glycol and paraffin etc.The lubricant that is used for these formulations comprises sodium oleate, sodium stearate, Magnesium Stearate, Sodium Benzoate, sodium acetate and sodium-chlor etc.Disintegrating agent includes but not limited to starch, methylcellulose gum, agar, bentonite and xanthan gum etc.When requiring aqeous suspension to orally use, activeconstituents and emulsifying agent and suspension agent are used in combination.If desired, can add some sweeting agent and/or sweetener.For intramuscular, intraperitoneal, subcutaneous and intravenously use, prepare the sterile liquid of activeconstituents usually, and should carry out suitable adjusting and buffering the pH value of solution.Use for intravenously, should control, so that make preparation etc. ooze the total concn of solute.
The compounds of this invention can also carry out administration with the form of liposome delivery system, and the liposome delivery system for example is little individual layer capsule, big individual layer capsule and multilayer capsule.Liposome can be by multiple phosphatide, and for example cholesterol, stearylamine or Yelkin TTS form.
The compounds of this invention also can be sent as individual carrier by utilizing monoclonal antibody, and wherein compound molecule is coupled on the monoclonal antibody.The compounds of this invention also can with the soluble polymer coupling as the drug target carrier.Above-mentioned polymkeric substance can comprise polyoxyethylene-polylysine that polyvinylpyrrolidone, pyran co-polymer, poly-hydroxy propyl methyl acid amides-phenol, poly-hydroxy-ethyl asparagine-phenol or palmitoyl replace.In addition, The compounds of this invention can be coupled to a class in reaching the controlled release of medicine on the useful biodegradable polymkeric substance, and biodegradable polymkeric substance for example is multipolymer, poly-epsilon-caprolactone, multi-hydroxybutyrate, poe, polyacetal, poly-dihydropyrane, polybutylcyanoacrylate and the crosslinked or amphipathic hydrogel segmented copolymer of poly-lactic acid, polyglycolic acid, poly-lactic acid and polyglycolic acid.
The compounds of this invention also can be used for and the known reagent coupling that is used for the treatment of or prevents following disease, and described disease comprises: osteoporosis, the osteoporosis that the suprarenal gland glucocorticosteroid brings out, osteitis deformans, abnormal bone metabolism raises, periodontopathy, the tooth infringement, fracture, rheumatic arthritis, osteoarthritis, the Periprosthetic osteolysis, the fragilitas ossium disease, obesity, atherosclerosis, chronic obstructive pulmonary disease, teenager's diabetes of showing effect, multiple sclerosis, chronic pemphigus, hyperthyroidism, myasthenia gravis, systemic lupus erythematosus, rheumatoid arthritis and struma lymphomatosa, asthma, exogenous immune response, parasitic infection, cancer, metastatic bone disease, malignant hypercalcemia or multiple myeloma.Compound disclosed by the invention and other are included in the scope of the present invention equally to the combination of treatment or preventing osteoporosis disease or other bone disorders useful reagent.Those of ordinary skills can distinguish which agent combination will be useful, and this is based on the characteristic of medicine and related disease.Mentioned reagent comprises following reagent: organic bisphosphonate, estrogenic agents, androgen receptor modifier, osteoclast proton Triphosaden enzyme inhibitors, HMG-CoA reductase inhibitor, integrain receptor antagaonists, scleroblast anabolic agent (for example PTH) and pharmacy acceptable salt and composition thereof.Preferably be combined as The compounds of this invention and organic bisphosphonate.Another preferably is combined as The compounds of this invention and estrogenic agents.Another preferably is combined as The compounds of this invention and androgen receptor modifier.Another preferably is combined as The compounds of this invention and scleroblast anabolic agent.
" organic bisphosphonate " includes but not limited to the compound of following chemical formula:
Figure A20048002452000241
Wherein n is 0～7 integer, and wherein A and X are independently selected from: H, OH, halogen, NH 2, SH, phenyl, C 1-C 30Alkyl, C 3-C 30Branched-chain alkyl or cycloalkyl, the twin nuclei that contains two or three N, C 1-C 30Substituted alkyl, C 1-C 10The NH that alkyl replaces 2, C 3-C 10The NH of branched-chain alkyl or cycloalkyl substituted 2, C 1-C 10The NH that dialkyl group replaces 2, C 1-C 10Alkoxyl group, C 1-C 10Sulfenyl, thienyl, halogeno-benzene sulfenyl, C that alkyl replaces 1-C 10Phenyl, pyridyl, furyl, pyrrolidyl, imidazolyl, imidazopyridyl and benzyl that alkyl replaces make that A and X are not selected from H or OH when n is 0; Perhaps A forms C with X altogether with carbon atom or with the atom that they link to each other 3-C 10Ring.
In above-mentioned chemical formula, alkyl can be straight chain, side chain or cyclic alkyl, is condition to select competent atom to be used for chemical formula.C 1-C 30Substituted alkyl can comprise multiple substituting group, and substituent limiting examples comprises following substituting group, is selected from: phenyl, pyridyl, furyl, pyrrolidyl, imidazoles ketone group, NH 2, C 1-C 10The NH that alkyl replaces or dialkyl group replaces 2, OH, SH and C 1-C 10Alkoxyl group.
For A and/or X substituting group, above-mentioned chemical formula also comprises complicated carbocyclic ring structure, aromatic structure and heteroatoms structure, and the limiting examples of these structures comprises naphthyl, quinolyl, isoquinolyl, adamantyl and chlorinated benzene sulfenyl.
The pharmacy acceptable salt of bisphosphonate and bisphosphonate derivative also are effective at this.The limiting examples of these salt comprises following salt, is selected from: an alkali metal salt, alkaline earth salt (alkaline metal), ammonium salt and single-, two-, three-or four-C 1-C 30The ammonium salt that alkyl replaces.Preferred salt is selected from sodium salt, sylvite, calcium salt, magnesium salts and ammonium salt.More preferably sodium salt.The limiting examples of derivative comprises and is selected from: the derivative of ester class, hydrate and acid amides.
Should be noted that at this to relate to the term " bisphosphonate " that uses in the therapeutical agent of the present invention and the implication of " bisphosphonate class " also comprises bisphosphonate, two phosphonic acids and di 2 ethylhexyl phosphonic acid, and the salt of these materials and derivative.Unless offer some clarification on, the use of the concrete nomenclature in relating to bisphosphonate does not also mean that limitation of the scope of the invention.Because skilled those of ordinary skill current application is mixed nomenclature, so, be that active weight with acid is a basic calculation otherwise relate to the specified weight of bisphosphonate compound of the present invention or per-cent unless explanation is arranged in addition at other place of this paper.For example, phrase " suppresses bisphosphonate for the about 5mg bone resorption based on the clinic effect of alendronate active weight, is selected from clinic effect of alendronate ester, its pharmacy acceptable salt and composition thereof " to be meant that the amount of the bisphosphonate compound of selection is that benchmark calculates with the 5mg clinic effect of alendronate.
Limiting examples at this effective bisphosphonate comprises following: Alendronate (having another name called clinic effect of alendronate), alendronate sodium, clinic effect of alendronate sodium trihydrate or 4-amino-1-hydroxy butylidene-1,1-di 2 ethylhexyl phosphonic acid monosodium trihydrate compound.
Alendronate is described in United States Patent (USP) 4,922, and in 007, people such as Kieczykowski are disclosed on May 1st, 1999; United States Patent (USP) 5,019,651, people such as Kieczykowski are disclosed on May 28th, 1991; United States Patent (USP) 5,510,517, people such as Dauer are disclosed on April 23rd, 1996; United States Patent (USP) 5,648,491, people such as Dauer are disclosed on July 15th, 1997, and their full text is hereby incorporated by.
Suberyl aminomethylene-1, the 1-di 2 ethylhexyl phosphonic acid, YM 175, Yamanouchi (she blocks phosphonic acids, is called cimadronate in the past) is as people's such as Isomura the United States Patent (USP) 4 that is disclosed in November 13 nineteen ninety, 970,335 is described, and it is hereby incorporated by in full.
1,1-dichloro methylene radical-1,1-bisphosphate (clodronic acid) and disodium salt (clodronate, Procter and Gamble), be described in belgian patent 672,205 (1966) and J.Org.Chem, in 32,4111 (1967), their full text of two all is incorporated herein by reference at this.
1-hydroxyl-3-(1-pyrrolidyl)-propylidene-1,1-di 2 ethylhexyl phosphonic acid (EB-1053).
1-hydroxyl ethane-1,1-bisphosphate (etidronic acid).
1-hydroxyl-3-(N-methyl-N-amyl group amino) propylidene-1, the 1-di 2 ethylhexyl phosphonic acid has another name called BM-210955, Boehringer-Mannheim (Ibandronic acid) is described in laid-open U.S. Patents numbering 4,927 on May 22 nineteen ninety, in 814, it is hereby incorporated by in full.
1-hydroxyl-2-imidazo (1,2-a) pyridin-3-yl ethylidene (minodronic acid).
6-amino-1-hydroxyl hexylidene-1,1-di 2 ethylhexyl phosphonic acid (neridronic acid).
3-(dimethylamino)-1-hydroxy propylidene-1,1-di 2 ethylhexyl phosphonic acid (Olpadronic acid).
3-amino-1-hydroxy propylidene-1,1-di 2 ethylhexyl phosphonic acid (pamidronic acid).
[[2-(2-pyridyl) ethylidene]-1,1-di 2 ethylhexyl phosphonic acid (piridronic acid) are described in the United States Patent (USP) numbering 4,761,406, and it is hereby incorporated by in full.
1-hydroxyl-2-(3-pyridyl)-ethylidene-1,1-di 2 ethylhexyl phosphonic acid (risedronic acid).
(4-chloro-phenyl-) sulphur methane-1,1-di 2 ethylhexyl phosphonic acid (tiludronic acid), as United States Patent (USP) 4,876, described in 248, it is hereby incorporated by in full.
1-hydroxyl-2-(1H-imidazoles-1-yl)-ethylidene-1,1-di 2 ethylhexyl phosphonic acid (Zoledronic acid).
The limiting examples of diphosphonate comprises clinic effect of alendronate, cimadronate, clodronic acid, etidronic acid, Ibandronic acid, she blocks phosphonic acids, minodronic acid, neridronic acid, Olpadronic acid, pamidronic acid, piridronic acid, risedronic acid, tiludronic acid and Zoledronic acid, and pharmacy acceptable salt or ester class.Especially preferred diphosphonate is an Alendronate, particularly the sodium of clinic effect of alendronate, potassium, calcium, magnesium or ammonium salt.The concrete example of preferred bisphosphonate is the clinic effect of alendronate sodium salt, particularly hydration clinic effect of alendronate sodium salt.Salt can carry out hydration with the water of whole mole number or the water of non-whole mole number.Another concrete example of preferred bisphosphonate is a hydration clinic effect of alendronate sodium salt, particularly when salt hydrate is clinic effect of alendronate monosodium trihydrate compound.
Should approve, can use the mixture of two or more bisphosphonate active substances.
The exact dosage desired of organic bisphosphonate will change with following condition, the character and severity and other relevant medical treatment and physical factor of the illness that comprise the concrete bisphosphonate, Mammals of dosage regimen, selection or human age, sex and state, will treat.Therefore, accurate pharmacy effective dose can not be predesignated, and can be determined immediately by care-giver or clinician.Suitable amount can be determined by routine test according to animal model and human clinical study.Usually, the sufficient quantity of bisphosphonate is selected to obtain the bone resorption restraining effect, i.e. the bisphosphonate of administration bone resorption amount of suppression.For the mankind, effective oral dosage of bisphosphonate is generally about 1.5～about 6000 μ g/kg body weight and preferred about 10～about 2000 μ g/kg body weight.For clinic effect of alendronate monosodium trihydrate compound, general human dosage is generally about 2 mg/day～about 40 mg/day, is preferably about 5 mg/day～about 40 mg/day.For clinic effect of alendronate monosodium trihydrate compound, U.S.'s dosage that is used for preventing osteoporosis disease of approval at present is 5 mg/day, and the dosage that is used for the treatment of osteoporosis is 10 mg/day, and the dosage that is used for the treatment of Paget's disease is 40 mg/day.
In another kind of dosage regimen, bisphosphonate administration at set intervals rather than by a day administration, for example weekly administration, twice administration weekly, per two all administrations and twice administration in every month.In weekly dosage regimen, clinic effect of alendronate monosodium trihydrate compound will be by the dosed administration in 35 milligrams/week or 70 milligrams/week.
" selective estrogen receptor modulators " is meant and do not consider mechanism, can disturb or suppress the compound that oestrogenic hormon is bonded to acceptor.The example of estrogenic agents including but not limited to oestrogenic hormon, progestogen, estradiol,
Figure A20048002452000271
Lip river former times sweet smell, raloxifene, Lasofoxifene, TSE-424, tamoxifen, idoxifene, LY353381, LY117081, toremifene, fulvestrant, 4-[7-(2,2-dimethyl-1-oxopropoxy-4-methyl-2-[4-[2-(piperidino) oxyethyl group] phenyl]-2H-1-chromene-3-yl]-phenyl-2,2-dimethyl propylene acid esters, 4,4 '-dihydroxy benaophenonel-2,4-dinitrophenyl-hydrazone and SH646.
" erss conditioning agent " is the compound (the exciting ER of ER promotes tryptophan hydroxylase gene transcription (TPH, the key enzyme during thrombotonin is synthetic) through ER mediation) of a kind of optionally excitement or anti-exciting erss.The example of estrogen receptor beta-agonists can obtain in open in that following PCT is international, is disclosed in the WO01/82923 in November 8 calendar year 2001 and is disclosed in the WO 02/41835 on May 20th, 2002, and their full text all is hereby incorporated by.
" androgen receptor modifier " is meant and do not consider mechanism, and interference or inhibition male sex hormone are bonded to the compound of acceptor.The example of androgen receptor modifier comprises finasteride and other 5 inhibitor, Nilutamide, Drogenil, bicalutamide, liarozole and acetate Abiraterone.
" osteoclast proton Triphosaden enzyme inhibitors " is meant the inhibitor of proton apysase, is found on the teleblem of osteoclast, and it is reported, it plays significant effect in the bone resorption process.This proton pump is represented the tempting target of bone resorption inhibitor design, and these inhibitor may and prevent effectively osteoporosis and associated metabolic treatment of diseases.Consult people such as C.Farina, " Selective inhibitors of the osteoclast vacuolarproton ATPase as novel bone antiresorptive agents ", DDT, 4:163-172 (1999), it is hereby incorporated by in full.
" HMG-CoA reductase inhibitor " is meant 3-hydroxy-3-methyl glutaryl-CoA reductase enzyme.The HMG-CoA reductase enzyme is had the active compound of inhibition can easily be obtained identifying by well-known assay method in the use prior art.For example, consult United States Patent (USP) 4,231, the assay method of describing or enumerating among the 30th～33 page of 938 the 6th hurdles and the WO 84/02131.When this uses, term " HMG-CoA reductase inhibitor " has identical implication with " inhibitor of HMG-CoA reductase enzyme ".
The example of adaptable HMG-CoA reductase inhibitor includes but not limited to lovastatin (MEVACOR , consult U.S. Patent number 4,231,938,4,294,926 and 4,319,039), Simvastatin (ZOCOR , consult U.S. Patent number 4,444,784,4820,850 and 4,916,239), Pravastatin (PRAVACHOL , consult U.S. Patent number 4,346,227,4,537,859,4,410,629,5,030,447 and 5,180,589), fluvastatin (LESCOL , consult U.S. Patent number 5,354,772,4,911,165,4,929,437,5,189,164,5,118,853,5,290,946 and 5,356,896), atorvastatin (LIP1TOR , consult U.S. Patent number 5,273,995,4,681,893,5,489,691 and 5,342,952) and Cerivastatin (also claim rivastatin and BAYCHOL , consult U.S. Patent number 5,177,080).The structural formula of these compounds and can be used for the HMG-CoA reductase inhibitor of the inventive method in addition as described below, M.Yalpani, " Cholesterol Lowering Drugs ", 85～89 pages the 87th page, on February 5th, 1996 and U.S. Patent number 4,782,084 and 4,885,314.Term HMG-CoA reductase inhibitor comprises all pharmaceutically acceptable lactones and open acid form (promptly wherein lactonic ring is opened and formed free acid) as used herein, and have the salt of compound of HMG-CoA reductase active and ester-formin and therefore the purposes of above-mentioned salt, ester, open acid and lactone form all be included in the scope of the present invention.Shown in the following structure I of example and II of lactone part and corresponding open acid form thereof.
Figure A20048002452000281
Lactone open loop acid
I II
In the HMG-CoA reductase inhibitor that can have open loop acid form, salt form and ester-formin can preferably be formed by open loop acid, and all these forms is included in the implication of term " HMG-CoA reductase inhibitor " as used herein.Preferred HMG-CoA reductase inhibitor is selected from lovastatin and Simvastatin and Simvastatin most preferably.At this, term " pharmacy acceptable salt " about the HMG-CoA reductase inhibitor will mean the non-toxic salt that is used for The compounds of this invention, they carry out prepared in reaction by free acid and suitable organic or mineral alkali usually, especially those salt that form by positively charged ion, positively charged ion is sodium for example, potassium, aluminium, calcium, lithium, magnesium, zinc and tetramethylammonium, and the salt that forms by amine, amine is ammonia for example, quadrol, N-methyl glucoside amine, Methionin, arginine, ornithine, choline, N, N '-dibenzyl-ethylenediamin, chloroprocaine, diethanolamine, PROCAINE HCL, PHARMA GRADE, the N-benzyl-1-phenylethylamine, 1-p-chlorobenzyl-2-Pyrrolidine-1 '-Ji-tolimidazole, diethylamine, piperazine and Tutofusin tris.The further example of the salt form of HMG-CoA reductase inhibitor can be including but not limited to, acetate; the benzene sulfonate benzoate; supercarbonate; hydrosulfate; the Tartaric acid hydrogen salt; borate; bromide; Ca-EDTA salt; d-camphorsulfonic acid; carbonate; muriate; clavulanate (clavulanate); Citrate trianion; dihydrochloride; edetate; ethanedisulphonate; estolate; esilate; fumarate; gluceptate; gluconate; glutaminate; the glycoloyl arsanilate; hexylresorcin salt (hexylresorcinate); breathe out amine (hydrabamine); hydrobromate; hydrochloride; Hydroxynaphthoate; iodide; different thiosulphate; lactic acid salt; Lactobionate (lactobionate); lauroleate; malate; maleate; mandelate; mesylate; methyl-sulfate; mucate (mucate); naphthalenesulfonate; nitrate; oleate; oxalate; pamoate (pamaote); palmitate; pantothenate (panthothenate); phosphate/phosphor acid hydrogen salt; polygalacturonate; salicylate; stearate; subacetate; succinate; tannate; tartrate; teoclate; tosylate; triethyl iodate thing and valerate.
The ester derivative of described HMG-CoA reductase inhibiter compounds can be used as prodrug, when it being absorbed when going in the warm blooded animal blood, can split in a certain way to discharge medicament forms and to make medicine that the therapeutic efficacy of improvement is provided.
As used above, " integrain receptor antagaonists " is meant selectivity antagonism, inhibition or offsets the compound that the physiology part is bonded to α v β 3 integrins, be meant selectivity antagonism, inhibition or offset the compound that the physiology part is bonded to α v β 5 integrins, be meant selectivity antagonism, inhibition or offset the compound that the physiology part is bonded to the compound of α v β 3 integrins and α v β 5 integrins and is meant the specific integrin that antagonism, inhibition or counteracting are expressed on capillary endothelial cell.This term also relates to α v β 6, α v β 8, α 1 β 1, α 2 β 1, α 5 β 1, α 6 β 1 and α 6 beta 1 integrin antagonists.Term also relates to the antagonist of the arbitrary combination of α v β 3, α v β 5, α v β 6, α v β 8, α 1 β 1, α 2 β 1 α 5 β 1, α 6 β 1 and alpha 6 beta 4 integrin.People such as H.N.Lode, PNAS USA, 96:1591-1596,1999, in the elimination that spontaneous tumor shifts, observed the synergistic effect between blood vessel originality alpha v integrin antagonist and tumor specific antibody cytokine (interleukin II) fusion rotein.Their result shows that this coupling is effective for the treatment of cancer and metastatic tumo(u)r growth.α v β 3 integrain receptor antagaonists suppress bone resorption by the new mechanism different with all existing medicines.Integrin is the transmembrane adhesion receptor of the allos dimerization of mediated cell-cell and cell-substrate interaction.α and β integrin subunit's noncovalent interaction and in divalent cation dependency mode in conjunction with extracellular substrate part.The integrin of maximum is α v β 3 (＞107/ osteoclast) on the osteoclast, and they appear to play a part speed limit in cytoskeletal organization, and cytoskeletal organization on cell migration and polarization are important.α v β 3 antagonistic actions are selected from bone resorption restraining effect, restenosis restraining effect, macular degeneration restraining effect, sacroiliitis restraining effect and cancer and metastatic tumo(u)r restraining effect.
" scleroblast anabolic agent " is meant the preparation of structure bone, for example Rat parathyroid hormone 1-34 (PTH).Shown at animal and human's class discontinuous administration Rat parathyroid hormone 1-34 (PTH) or its N-terminal fragment and analogue thereof, can prevent, stop, partly reverse the bone loss and stimulate osteogenesis.Detailed description relates to people such as D.W.Dempster, " Anabolicactionsof parathyroid hormone on bone ", Endocr Rev, 14:690-709,1993.Thereby research has shown the clinical benefit of Rat parathyroid hormone 1-34 in stimulating osteogenesis and raising bone mass and intensity.The result is by people such as RM Neer report, New Eng J Med, 344:1434-1441,2001.
In addition, parathyroid hormone-related protein matter fragment or its analogue, for example PTHrP-(1-36) has shown that having the effect of effective anticalcium urine [consults people such as M.A.Syed, " Parathyroid hormone-related protein-(1-36) stimulatesrenal tubular calcium reabsorption in normal human volunteers:implications for the pathogenesis of humoral hypercalcemia ofmalignancy ", JCEM, 86:1525-1531 (2001)], and also can be used as the anabolic agent for the treatment of osteoporosis.
If be mixed with fixed dosage, this combined prod uses The compounds of this invention and other forms of pharmacologically active agents in its approval dosage range in the dosage range as described below.When combination preparation was not suitable for, The compounds of this invention can use in order with known pharmaceutically acceptable reagent in addition.
Term relevant with The compounds of this invention " administration " and distortion (for example, " medication " compound) thereof are meant that the prodrug with compound or compound is incorporated in the animal system that needs treatment.When The compounds of this invention or its prodrug with in one or more other promoting agents (for example cytotoxic agent etc.) when combination provides, " administration " and distortion thereof all be interpreted as inclusion compound or its prodrug and other reagent and stream is introduced and order is introduced.The prodrug that comprises The compounds of this invention within the scope of the present invention.Usually, prodrug is the functional derivatives of The compounds of this invention, is easy to convert to the compound that needs in vivo.Therefore, in methods of treatment of the present invention, term " administration " should comprise the treatment of multiple described state, and using clear and definite disclosed compound or use can clear and definite disclosed compound, but behind the administration patient, this compound changes into specified compound in vivo.Suitably the selection of prodrug derivatives and the conventional steps of preparation are described in, for example, " Design of Prodrugs ", chief editor H.Bundgaard, Elsevier, 1985, it is hereby incorporated by in full.The meta-bolites of these compounds comprises the active centre that is produced when The compounds of this invention is introduced coenocorrelation.
The term of Shi Yonging " composition " comprises the product of the appointment composition that comprises specified amount in this article, and any directly or indirectly by the product that branch combination obtains that is designated as of specified amount.
Term used herein " treatment significant quantity " is meant the amount of active substance or pharmaceutical preparation, this amount can cause in tissue, system, animal or the mankind that biological respinse or drug reaction, this reaction are the reactions that researchist, animal doctor, medical doctor or other clinician look for.
" treatment " of term disease used herein comprises: preventing disease, and promptly cause the clinical symptom of disease in Mammals, not develop, Mammals may be exposed to disease or easy infection disease, but does not experience again or the demonstration disease symptoms; Suppress disease, promptly stop or reduce the development of disease or its clinical symptom; Perhaps palliate a disease, promptly cause disease or its clinical symptom to disappear.
The term of Shi Yonging " bone resorption " is meant a process in this article, through this process osteoclast degraded bone.
The present invention also comprises the pharmaceutical composition useful to the treatment of osteoporosis or other bone disorders, comprises the The compounds of this invention of drug treatment significant quantity, contains or do not contain pharmaceutically acceptable carrier or thinner.Suitable compositions of the present invention comprises the aqueous solution that comprises acceptable carrier on The compounds of this invention and the pharmacology, salt solution for example, and its pH value level for example is 7.4.Solution can be incorporated in the blood samples of patients by local bolus injection.
When compound administration according to the present invention is entered the man-hour that is put to the test, every day, dosage was determined by the doctor that writes a prescription usually, and dosage is different according to the severity of age, body weight and the reaction of individual patient and patient's symptom usually simultaneously.
In an example use, with the compound administration of sufficient quantity to the Mammals that the illness that depends on kethepsin is treated.Oral dosage of the present invention when being used for producing needed effect, is about 0.01 mg/kg body weight/day (mg/kg/ days)～about 100mg/kg/day, preferred 0.01～10mg/kg/day and 0.1～5.0mg/kg/day most preferably.For oral administration, composition preferably provides with tablet form, wherein contain 0.01,0.05,0.1,0.5,1.0,2.5,5.0,10.0,15.0,25.0,50.0,100 and 500 milligram of activeconstituents, so that adjustment is administered to the patient's who treats dosage according to symptom.Medicament generally contains the 0.01mg that has an appointment～about 500mg activeconstituents, preferably about 1mg～about 100mg activeconstituents.For intravenous administration, during the constant speed transfusion, most preferred dosage is about 0.1～about 10mg/kg/min.Advantageously, The compounds of this invention can be with single per daily dose administration, and perhaps every day, total dose can be divided into every day twice, three times or four dosed administrations.In addition, for The compounds of this invention, preferably can be with form administration in the nose, carrier in suitable nose is used in the part is perhaps through seeing through skin route, the skin surface patch form of utilizing those those of ordinary skills to know.To see through skin delivery system form administration, dosed administration will continue whole dosage regimen certainly, rather than dosed administration intermittently.
The compounds of this invention can use with other agent combination that is used for the treatment of the state of kethepsin mediation.The single component of aforesaid combination can be divided into different number of times administrations during treating, perhaps with separately or the parallel administration of the form of single combination.Therefore the present invention is interpreted as comprising the scheme of all above-mentioned combination therapys or wheel stream treatment, and term " administration " should correspondingly be understood.Should be appreciated that The compounds of this invention and other can be used for the scope of combination of agents of the illness of treated tissue proteolytic enzyme mediation, comprise any and the combination that can be used for treating with the pharmaceutical composition of estrogen function associated conditions in principle.
Therefore, the scope of the invention comprises the compound of requirement of the present invention and the purposes of second agent combination, and second reagent is selected from: organic bisphosphonate, estrogenic agents, androgen receptor modifier, osteoclast proton Triphosaden enzyme inhibitors, HMG-CoA reductase inhibitor, integrain receptor antagaonists, scleroblast anabolic agent (for example PTH) and pharmacy acceptable salt and composition thereof.
According to the instruction that is included in herein, these and other aspect of the present invention will be conspicuous.
Replenish the above, the detailed description of one or more embodiments of the invention is set forth.Though it is any to described similar or equivalent method and material can be used for the present invention's practice or test, preferred present described method and material herein.According to specification sheets with according to claims, other features, objects and advantages of the present invention will be conspicuous.In specification sheets and appended claims, unless context clearly indicates, otherwise singulative comprises a plurality of objects.Unless otherwise defined, technical term that all use in this article and scientific terminology have and the identical implication of the technical field of the invention those of ordinary skill common sense.Patent and publication that all are quoted in this manual all are incorporated herein by reference.
Above-mentioned specification sheets only is to exist for illustrative purposes, does not plan the present invention is defined as clear and definite disclosed form, but limits by the claim that is additional to this.
Scheme
The compounds of this invention can obtain preparation according to scheme 1, and is as follows.The alpha-amino group ester can be joined in the haloalkane ketone thus and form aminal, the aminal formation imines that in the presence of dewatering agent, can dewater, dewatering agent for example is TiCl 4, MgSO 4Perhaps sec.-propyl trifluoroacetate.Form amine with reductive agent (for example sodium cyanoborohydride or sodium borohydride) reduction imines.Ester hydrolysis and form acid amides with the aminoacetonitriles of suitable replacement provides The compounds of this invention.If the substituting group on the d system is a halogen, just provide additional compounds of the present invention with the suitable catalytic Suzuki coupling of boric acid generation palladium.In addition, carry out copper catalysis or the catalytic Buchwald coupling of palladium and additional compounds of the present invention is provided with suitable amine.In addition, the amine formation acid amides with suitable provides additional compounds of the present invention succeeded by palladium catalysis carboxylation.
Scheme 1
The compounds of this invention also can obtain preparation according to scheme 2, and is as follows.Ketone or aldehyde can carry out condensation with amino alcohol and produce the cyclic aminal.Handling with three normal Grignard reagents or organolithium reagent to provide suitable alkylating amino alcohol.With chromium system (for example Jones oxidation or H 5IO 6/ CrO 3) oxidation alcohol, perhaps by the second oxidation effect (for example behind the NaClO, succeeded by oxalyl chloride/DMSO/Et 3N) oxidation alcohol will provide corresponding carboxylic acid.Carry out the peptide coupling and described Suzuki reaction will provide The compounds of this invention as scheme 1.
Scheme 2
Figure A20048002452000351
The compounds of this invention also can obtain preparation according to scheme 3, and is as follows.Ketone or aldehyde can carry out condensation with amino alcohol and produce acyclic aminal.Handling with many times of normal Grignard reagents or organolithium reagent to provide suitable alkylating amino alcohol.This alcohol can change into The compounds of this invention by the method for describing in the scheme 2.
Scheme 3
Figure A20048002452000361
The compounds of this invention also can obtain preparation according to scheme 4.Suitably the acetate that replaces can carry out enolization with suitable alkali (including but not limited to LDA, KHMDS, NaH or nBuLi) and handle to produce glycol with Paraformaldehyde 96.Use fluorination reagent (for example DAST) this glycol can be converted into difluoride.Curtius resets the back this ester is hydrolyzed, so will form amine.This amine can replace the α-bromo-ester of suitable replacement to form the alpha-amino group ester.The alpha-amino group ester can change into The compounds of this invention by the method for describing in the scheme 1.
Scheme 4
Figure A20048002452000371
The compounds of this invention also can obtain preparation according to scheme 5, and is as follows.Aldehyde or hemiacetal can carry out condensation azeotropic water removal simultaneously with amino alcohol, and alcohol moiety in the amino alcohol is protected with suitable protecting group.Handle the imines that produces with Grignard reagent or organolithium reagent suitable alkylating amino alcohol will be provided.Therefore the alcohol protecting group can be removed, and alcohol can change into The compounds of this invention, perhaps by the method described in the scheme 2, perhaps at first carries out the Suzuki reaction, succeeded by using H 5IO 6/ CrO 3Oxidation is pure and mild carries out the peptide coupling then.
Scheme 5
Figure A20048002452000381
The compounds of this invention also can obtain preparation according to scheme 6, and is as follows.The peptide coupling of a-amino acid is described in scheme 1,2 or 5, carries out coupling with the alpha-amino group acid amides, then to the primary amide that produces dewater (Voegel, J.J.; Benner, S.A., Helv.Chez.Aacta, 1996,79,1863) will form The compounds of this invention.
Scheme 6
The synthetic of some amino alcohols of using in scheme 2,3 and 5 originally is described in the scheme 7～11.For example, (2S)-synthesizing of 2-amino-4-fluoro-4-methylpent-1-alcohol in following scheme 7, obtain description, wherein R=Me.From suitable two protection aspartic acids, the document step of use standard can become carboxyl reduction alcohol, and (that is, acid anhydrides forms the back succeeded by using NaBH 4Reduction).2-amino-4-methylpentane-1, the protection form of 4-glycol (R=Me) can obtain generating then by suitable Grignard reaction or organic lithiation.At last, use fluorizating agent (for example DAST) hydroxylic moiety can be changed into the fluorine of expectation.The protection of this alcohol or unprotect form can change into The compounds of this invention according to scheme 1,2,3 and 5 then.
Scheme 7
4-fluoro leucine also can be synthesized according to scheme 8.With 4, the 5-leuceine changes into (4S)-4-(2-methyl-prop-2-thiazolinyl)-1,3- azoles alkane-2-ketone, and following scheme is described.Use hydrofluorination reagent (for example HF-pyridine) that this intermediate is handled then, produce (4S)-4-(2-fluoro-2-methyl-propyl)-1,3- azoles alkane-2-ketone.Carrying out alkaline hydrolysis then (is Ba (OH) 2Perhaps NaOH) produces (2S)-2-amino-4-fluoro-4-methylpentane-1-alcohol.
Scheme 8
4, the synthetic of 4-two fluoro-L-norvalines is described in the following scheme 9, wherein R=Me.Two protection Serines from suitable can use reagent (for example (PhO) 3P +MeI -) carry out iodate.
The zinc impregnation of synthetic iodide can use Zn-Cu to carrying out with TMSCl.Then the synthetic zincate can with alkane acyl chlorides generation palladium catalyzed coupling reaction, generate ketone.
At last, use fluorizating agent (for example DAST) ketone partly can be changed into the difluoro derivatives of expectation.Then, the protection of this amino acid or amino alcohol or unprotect form can change into The compounds of this invention according to scheme 1,2,3 and 5.
Scheme 9
Figure A20048002452000421
Being used for amino alcohol of the present invention can also be synthesized according to scheme 10.With the amino acid of band protecting group with reductive agent (NaBH for example 4) reduction, there is or do not have auxiliary agent (for example LiCl), in a kind of solvent (for example EtOH) or mixed solvent system (for example EtOH and THF), carry out.According to the character of protecting group, amino protecting group is removed with appropriate means then.
Scheme 10
Figure A20048002452000431
Be used for that of the present invention (2S, 4S)-2-amino-5,5,5-three the synthetic of fluoro-4-methylpent-1-alcohol are described in the scheme 11.N-benzoyl-5,5, and 5-trifluoro leucine (people such as Ojiima, J.Org.Chem., 1989,54,4511-4522) can under refluxad be hydrolyzed with aqueous acid (for example 6MHCl).Then the amino acid salts hydrochlorate is changed into N-ethanoyl-5,5,5-trifluoro leucine, and amino chiral centre by enzyme catalysis method obtain splitting (Synthetic Communications, 1996,26,1109-1115).To isolating 5,5,5-three fluoro-L-leucines are protected with protecting group (for example benzyl carbamate) and carboxyl are carried out esterification then.Two non-corresponding isomer in the 4-position obtain separating by flash column chromatography then.To wherein a kind of enantiomer then, (2S, 4S) Bao Hu amino acid changes into amino alcohol, shown in scheme 10.
Scheme 11
Figure A20048002452000441
R wherein 5Be hydrogen and R 6The The compounds of this invention that is aryl or heteroaryl also can obtain preparation according to scheme 12, and is as follows.Aryl or heteroaryl aldehyde with suitable protecting group the amino alcohol that alcohol moiety has wherein carried out protection is carried out condensation; with Grignard reagent or organolithium reagent the imines that produces is handled then, Grignard or organolithium reagent are chemical formula halo-(D) n-Li or halo-(D) n-MgX (wherein defining in D such as the summary of the invention) forms alkylating amino alcohol succeeded by removing the deoxidation protecting group.Then alkylating amino alcohol is changed into The compounds of this invention, perhaps by the method described in the scheme 2, perhaps by at first with formula R 7-B (OH) 2Boric acid ester carry out Suzuki reaction, then with suitable oxygenant (H for example 5IO 6/ CrO 3) oxidation alcohol and produce acid and last under peptide coupling condition as discussed previously, with aminoacetonitriles acid is handled.
Scheme 12
The compounds of this invention also can obtain preparation according to scheme 13, and is as follows.In the presence of sodium iodide; in suitable organic solvent (for example dimethyl formamide); suitably the amino acid derivative and the trimethylene oxide tosylate of N-protected react, and form corresponding oxetane, oxetane are handled forming ortho ester with diborane.Remove amino protecting group and produce amine, amine and formula R 6CHO (R wherein 6Be aryl or heteroaryl) aldehyde or with formula R 6Condensation is taking place under the reaction conditions in C (OH) (OR) hemiacetal of (wherein R is an alkyl) as mentioned above, forms imines.Under reaction conditions as mentioned above, handle imines and form the N-alkyl derivative with Grignard reagent or organolithium reagent.Remove ortho ester and form corresponding carboxylic acid, this carboxylic acid succeeded by carrying out Suzuki reaction as mentioned above, changes into The compounds of this invention by under peptide coupling condition condensation taking place with aminoacetonitriles then.
Scheme 13
Figure A20048002452000461
The compounds of this invention also can the method as shown in scheme 14 obtain preparation.Contain suitable R 1, R 2, R 2, R 4And R 6The aryl halide of group can produce aryl tetramethylethylene glycol thing with two (tetramethylethylene glycolization) two boron couplings.The latter can with R 7The coupling under the Suzuki condition of-bromide forms The compounds of this invention.
Scheme 14
Figure A20048002452000471
The compounds of this invention also can obtain preparation according to scheme 15, and is as follows.Amino acid and alpha-amino group ester, alcohol or the coupling of ketone generation peptide of the suitable replacement of describing in scheme 1,2 or 5 will form The compounds of this invention.Under the situation of α-An Jichun, oxygenated products alcohol will form the ketone as The compounds of this invention.Under the situation of alpha-amino group ester, the hydrolysis prods ester is succeeded by forming acid amides with the amine that suits, with the acid amides that forms as The compounds of this invention.
Scheme 15
Figure A20048002452000481
Acid shown in the scheme 1,2,6 and 15 also can obtain preparation shown in scheme 16.Suitably bromotoluene, benzyl iodide or the benzyl trifluoromethanesulfonic acid thing (can be chirality or racemic) that replaces can carry out coupling with the alpha-amino group ester under alkaline condition.The aqueous solution with alkali is hydrolyzed to it then, forms acid, and the latter can be converted into embodiments of the invention.
Scheme 16
Figure A20048002452000482
Following examples have been described the synthetic of selected The compounds of this invention.These embodiment are the concrete examples that require the invention of patent protection herein, and can not be interpreted as limitation of the scope of the invention.Table 1 has been described embodiment, when combining with the reactive site that is active kethepsin, is how to satisfy criterion distance as described herein for embodiment.
Embodiment 1
N 1-(cyanogen methyl)-N 2Synthesizing of-(2,2,2-three fluoro-1-phenylethyls)-L-leucine amide
Figure A20048002452000491
To L-leucine methyl ester hydrochloride (975mg in methylene dichloride 5.37mmol) (30mL) solution, adds 2,2, the 2-trifluoroacetophenone (0.75mL, 5.34mmol) and diisopropylethylamine (3.5mL, 20mmol).Drip and add the TiCl that is dissolved in the 0.45mL dichloromethane solution 4(0.55mL, 5.0mmol) and stir the mixture and spend the night.Add TiCl then in addition 4(O.4mL, 3.6mmol), and the 3h that stirs the mixture.Add NaCNBH 3(1050mg, methyl alcohol 16.7mmol) (20mL) solution, and the 2h that stirs the mixture.Pour among the 1N NaOH and and extract with ethyl acetate (2 *).Organic phase is used MgSO then with 1N NaOH and salt water washing 4Drying is also evaporated.Through ISCO column chromatography (30%～90% ethyl acetate/hexane gradient) purifying, form methyl N-(2,2,2-three fluoro-1-styroyls)-L-leucinate.
Methyl N under room temperature-(2,2,2-three fluoro-1-styroyls)-L-leucinate (150mg, 0.50mmol) 2: add 1M LiOH in the 1THF/MeOH solution.Stir the mixture whole night and concentrate.Resistates is distributed between the phosphate buffered saline buffer of ethyl acetate and pH value 3.5.With salt water washing organic phase, use MgSO 4Dry and concentrate, produce N-(2,2,2-three fluoro-1-phenylethyls)-L-leucine.
N-(2,2,2-three fluoro-1-phenylethyls)-L-leucine (149mg, 0.50mmol), the aminoacetonitriles hydrochloride (102mg, 1.1mmol) and PyBOP (260mg, 0.50mmol) mixture is dissolved among the DMF (5mL).(0.3mL 2.1mmol) and stir the mixture and spend the night, pours the pH value then into and is in 3 the phosphate buffered saline buffer, and extracts with 3: 1 ether/ethyl acetate to add triethylamine.With the saturated NaHCO of organic phase 3MgSO is used in the aqueous solution and salt water washing 4Drying is also evaporated.Through ISCO column chromatography (the ethyl acetate/hexane gradient is 20%～50%) purifying, form N 1-(cyanogen methyl)-N 2-(2,2,2-three fluoro-1-phenylethyls)-L-leucine amide is 1: 1 a non-corresponding isomer mixture.
MS(+APCI)：313.9[M+1]。
Embodiment 2
N 2-[1-(4-bromophenyl)-2,2,2-trifluoroethyl]-N 1Synthesizing of-(cyanogen methyl)-L-leucine amide
Figure A20048002452000501
Use the method for embodiment 1, prepare N 2～[1-(4-bromophenyl)-2,2,2-trifluoroethyl]-N 1-(cyanogen methyl)-L-leucine amide.
MS(-ESI)：403.9，405.9[M-1] -
Embodiment 3
N 1-(cyanogen methyl)-N 2Synthesizing of-{ [4 '-((methylsulfonyl))-1,1 '-xenyl-4-yl] [4-((methylsulfonyl)) phenyl] methyl }-L-leucine amide
Figure A20048002452000502
Step 1:N-{ (4-bromophenyl) [4-((methylsulfonyl)) phenyl] methylene radical }-L-leucine methyl esters
With (4-bromophenyl) [4-((methylsulfonyl)) phenyl] ketone (202mg, 0.59mmol), L-leucine methyl ester hydrochloride (328mg, 2.0mmol) and camphorsulfonic acid (52mg, the toluene solution of 0.22mmol use the Dean-Stark trap to reflux 18 hours.Except that desolvating and using EtOAc and hexane as elutriant, with chromatography the gained resistates is carried out purifying under vacuum, obtaining title compound is 1: 1 mixture with beginning material (4-bromophenyl) [4-((methylsulfonyl base)) phenyl] ketone ratio.
Step 2:N-{ (4-bromophenyl) [4-((methylsulfonyl base)) phenyl] methyl }-L-leucine methyl esters
Be 1: 1 N-{ (4-bromophenyl) [4-((methylsulfonyl)) phenyl] methylene radical to the ratio that derives from step 1 } leucine methyl esters and (4-bromophenyl) [4-((methylsulfonyl)) phenyl] ketone (185mg;～0.2mmol) acetate/the methyl alcohol (1: 3 of mixture; 4mL) in the solution; use the solid feed hopper add in batches sodium borohydride (～400mg), in two days, (stop night adding) and added once in per 30 minutes.Reaction mixture obtains distributing between EtOAc and water, uses Na 2SO 4Organic layer is carried out drying and concentrates.The gained mixture obtains purifying by chromatography, uses EtOAc and hexane as elutriant.Obtain N-{ (4-bromophenyl) [4-((methylsulfonyl)) phenyl] methyl }-L-leucine methyl esters, for no coloring agent and (4-bromophenyl) [4-((methylsulfonyl)) phenyl] methyl alcohol, be white solid.
Step 3:N-{ (4-bromophenyl) [4-((methylsulfonyl)) phenyl] methyl }-the L-leucine
To the N-{ that derives from step 2 (4-bromophenyl) [4-((methylsulfonyl)) phenyl] methyl }-L-leucine methyl esters (81mg, in THF 0.17mmol) (1mL) and MeOH (0.5mL) solution, adding 1NLiOH (0.3mL, 0.3mmol).At room temperature stir the gained mixture 18 hours, and between EtOAc and water, distributed then, add 1N HCl (0.5mL).With organic layer Na 2SO 4Drying is filtered and vacuum concentration, produces colourless gelationus title compound.
Step 4:N 2-{ (4-bromophenyl) [4-((methylsulfonyl)) phenyl] methyl }-N 1-(cyanogen methyl)-L-leucine amide
To N-{ (4-bromophenyl) [4-((methylsulfonyl)) phenyl] methyl that is cooled to-10 ℃ }-L-leucine (76mg; 0.17mmol) (derive from step 3), HATU (146mg; 0.38mmol) and aminoacetonitriles hydrochloride (52mg; 0.56mmol) DMF (1.1mL) solution in; add N; the N-diisopropylethylamine (0.13mL, 0.75mmol).Make reaction at room temperature carry out 18h and it is distributed between EtOAc and water.Organic layer Na 2SO 4Dry, filter and under vacuum, concentrate.Raw product uses EtOAc and hexane to carry out purifying as elutriant through chromatography, produces colourless gelationus title compound.
Step 5:N 1-(cyanogen methyl)-N 2-{ [4-((methylsulfonyl)) phenyl] [4 '-(methylthio group)-1,1 '-xenyl-4-yl] methyl }-L-leucine amide
Will be from the N of step 4 2-{ (4-bromophenyl) [4-((methylsulfonyl)) phenyl] methyl }-N '-(cyanogen methyl)-L-leucine amide (72mg; 0.15mmol) and 4-(methylthio group) phenyl-boron dihydroxide (37mg; 0.22mmol) the glycol dimethyl ether (1mL) and the multiphase mixture of 2M aqueous sodium carbonate under vacuum, outgas and use nitrogen wash.
The methylene dichloride title complex of adding [1,1 '-two (diphenyl phosphine) ferrocene] palladium chloride (II) in this mixture (19mg, 0.023mmol), succeeded by outgasing and using nitrogen purge.Reaction mixture was heated 16 hours at 85 ℃ under effectively stirring.With the NH of reaction mixture at EtOAc and 25%w/v 4Distribute between the OAc aqueous solution.Organic layer Na 2SO 4Dry, filter and under vacuum, concentrate.Raw product uses EtOAc and hexane to carry out purifying as elutriant through chromatography, produces colourless gelationus title compound.
Step 6:N 1-(cyanogen methyl)-N 2-{ [4 '-(methylthio group)-1,1 '-xenyl-4-yl] [4-((methylsulfonyl)) phenyl] methyl }-L-leucine amide
To N 1-(cyanogen methyl)-N 2-{ [4-((methylsulfonyl)) phenyl] [4 '-(methylthio group)-1; 1 '-xenyl-4-yl] methyl }-L-leucine amide (63mg; 0.12mmol), sodium tungstate dihydrate (2mg; 0.006mmol) and 4-butyl ammonium hydrogen sulfate (4mg; 0.01mmol) in the solution; (100 μ L 0.9mmol), at room temperature stir the mixture 10 minutes of gained to add the 30%w/v aqueous hydrogen peroxide solution.Reaction mixture distributes between EtOAc and water, adds 1MNaHSO 3(～3: 1).Use Na 2SO 4Dry organic layer, filtration and under vacuum, concentrate.Raw product uses EtOAc and hexane to carry out purifying as elutriant through chromatography, produces colourless gelationus title compound.
MS(+ESI)：568.2[M+1]+。
Embodiment 4
N 1-(cyanogen methyl)-N 2-{ (1S)-2,2,2-three fluoro-1-[4 '-((methylsulfonyl))-1,1 '-xenyl-4-yl] ethyl }-L-leucine amide synthetic
Figure A20048002452000531
Step 1:(2S)-and the 1-{[tertiary butyl (dimethyl) silyl] the oxygen base }-4-methylpentane-2-amine
Add triethylamine (11mL), DMAP (0.1g) and tert-butyldimethylsilyl chloride thing (8.5g) in L-leucinol (6.0g) methylene dichloride (100mL) solution under room temperature.At room temperature stirred the mixture 2 hours, and added entry then.Organic layer is separated, and further use the dichloromethane extraction aqueous solution.With the organic layer salt water washing that merges, with dried over mgso and under vacuum except that desolvating, obtain title compound, this resistates former state is used for next reaction.1H?NMR(CD 3COCD 3)δ3.48(m，2H)，3.32(m，1H)，2.76(m，1H)，1.78(m，1H)，1.22-1.02(m，2H)，0.88(m，15H)，0.06(s，6H)。
Step 2:(2S)-and the 1-{[tertiary butyl (dimethyl) silyl] the oxygen base }-the 4-methyl-N-[(1E)-2,2,2 trifluoro ethylidene] pentane-2-amine
Will be from (2S)-1-{[tertiary butyl (dimethyl) silyl of step 1] the oxygen base }-toluene (300mL) vlil of 4-methylpent-2-amine (50g) and trifluoro acetaldehyde methyl hemiacetal (35mL) 16 hours, in the Dean-Stark trap, collect water during this period.The vaporising under vacuum solvent and with resistates at SiO 2On carry out purifying, use hexane and ethyl acetate (9: 1) as elutriant, obtain title compound.
1H?NMR(CD 3COCD 3)δ7.88(m，1H)，3.76-3.45(m，3H)，1.60-1.25(m，3H)，0.88(m，15H)，0.06(s，3H)，0.04(s，3H).
Step 3:(2S)-and 2-{ (1S)-1-(4-bromophenyl)-2,2, the 2-trifluoroethyl] amino }-4-methylpentane-1-alcohol
With n-BuLi (the 2.5M hexane solution, 42mL) join-70 ℃ 1, in the THF solution of 4-dibromobenzene (25.8g), and stirred the mixture 25 minutes.Add (2S)-1-{[tertiary butyl (dimethyl) silyl then] the oxygen base }-4-methyl-N-[(1E)-2,2,2-trifluoro ethylidene] THF (30mL) solution of pentane-2-amine (31g), and stirred the mixture 1.5 hours.Under violent stirring, it is slowly poured in the mixture of ethyl acetate (500mL), water (2L), ice (300g) and ammonium chloride (100g) then.Organic layer is separated, and (2 * 500mL) extract the aqueous solution further to use ethyl acetate.The organic layer salt water washing that merges obtains resistates with dried over mgso with under vacuum except that desolvating, and this resistates uses like this.To be dissolved among the THF (250mL) by last gained resistates, and solution will be cooled to 0 ℃.The 1MTHF solution of tertiary butyl Neutral ammonium fluoride (110mL) is dripped adding, and made mixture reaction 4 hours.Under violent stirring, be poured in ethyl acetate (300mL), water (2L) and the ammonium chloride (100g).Organic layer is separated, and (2 * 100mL) aqueous layer extracted of further using ethyl acetate.Organic layer salt water washing with merging obtains resistates with dried over mgso and vaporising under vacuum solvent, with this resistates at SiO 2On carry out purifying, use gradient ethyl acetate and hexane (1: 5～1: 4) elutriant, obtain title compound.
1H?NMR(CD 3COCD 3)δ7.6(2H，d)，7.45(2H，d)，4.55(1H，m)，3.65-3.7(1H，m)，3.5-3.55(1H，m)，3.25-3.35(1H，m)，2.6-2.7(1H，m)，2.25-2.35(1H，m)，1.65-1.75(1H，m)，1.3-1.4(1H，m)，1.2-1.3(1H，m)，0.75-0.9(6H，dd).
Step 4:(2S)-4-methyl-2-((1S)-2,2,2-three fluoro-1-[4 '-(methylthio group)-1,1 '-xenyl-4-yl] and ethyl } amino) pentane-1-alcohol
Nitrogen gas stream was passed through suspension 15 minutes, and this suspension is by bromide (27.7g), 4-(methylthio group) phenyl-boron dihydroxide (15.7g), 2M Na from step 3 2CO 3(100mL) and n-propyl alcohol (500mL) form.Add Pd (OAc) then 2And PPh 3Ratio is 1: 3 mixture (3.5g) and reaction is warming up to 70 ℃ and stirred 8 hours under nitrogen.Mixture is cooled to room temperature, with ethyl acetate dilution and pour water (2L) into and ice in (500g).Ethyl acetate layer is separated, and further use ethyl acetate (200mL) aqueous layer extracted.With the acetic acid ethyl ester extract that merges with 0.5N NaOH (2 * 200mL), NH 4The Cl aqueous solution and salt water washing, and use dried over mgso.Removing desolvates stays resistates, at SiO 2By this resistates of chromatography purification, use gradient ethyl acetate and hexane (1: 4～1: 3) and use gradient acetone again and toluene (1: 10) wash-out.Resistates is dissolved in the hot hexane (200mL), and under agitation makes solution be cooled to 0 ℃.The solid that obtains is filtered and drying, obtain title compound.
1H?NMR(CD 3COCD 3)δ7.7(2H，d)，7.65(2H，d)，7.6(2H，d)，7.35(2H，d)，4.5-4.6(1H，m)，3.7(1H(OH)，m)，3.5-3.6(1H，m)，3.3-3.4(1H，m)，2.7(1H，m)，2.5(3H，s)，2.3-2.4(1H(NH)，m)，1.65-1.75(1H，m)，1.2-1.4(3H，m)，0.8-0.9(6H，dd).
Step 5:(2S)-4-methyl-2-((1S)-2,2,2-three fluoro-1-[4 '-((methylsulfonyl))-1,1 '-xenyl-4-yl] and ethyl } amino) pentane-1-alcohol
In toluene (400mL) solution of 0 ℃ sulfide (19g), add Na from step 4 2WO 42H 2O (0.16g) and Bu 4NHSO 4(0.81g).Slowly add 30% hydrogen peroxide (12.2mL) then, at room temperature stirred the mixture 4.5 hours.Then compound is poured on the mixture of ice, the sodium thiosulfate solution that dilutes and ethyl acetate.Organic layer is separated, and (2 * 100mL) aqueous layer extracted of further using ethyl acetate.Organic layer salt water washing with merging obtains resistates with dried over mgso and vaporising under vacuum solvent, with this resistates at SiO 2On carry out purifying, use ethyl acetate and hexane (1: 5～1: 4), to obtain product as elutriant.
1H?NMR(CD 3COCD 3)δ8.05(2H，d)，8.0(2H，d)，7.85(2H，d)，7.7(2H，d)，4.6-4.7(1H，m)，3.75(1H，m)，3.6(1H，m)，3.35-3.45(1H，m)，3.2(3H，s)，2.7-2.8(1H，m)，2.35-2.45(1H，m)，1.7-1.8(1H，m)，1.2-1.5(2H，m)，0.8-0.95(6H，dd).
Step 6:N-{ (1S)-2,2,2-three fluoro-1-[4 '-((methylsulfonyl))-1,1 '-xenyl-4-yl] ethyl }-the leucic preparation of L-
With H 5IO 6/ CrO 3Suspension (529mL 0.44M CH 3CN solution; Consult following note) be cooled to 0 ℃, dropping will be from the CH of the alcohol (20g) of step 3 3CN (230mL) solution.Under 0～5 ℃, stirred the mixture 3.5 hours.Under vigorous stirring, be poured into the pH value and be 4 Na 2HPO 4In and with ether (3 * 250mL) extract mixture.The ethereal extract water and the salt solution (1: 1) that merge are washed, use NaHSO 3Dilute solution and salt water washing.With the dried over sodium sulfate organic extract, filter and the solvent evaporate to dryness obtained resistates, resistates is divided into two groups to carry out following purifying.
To be dissolved in by the crude product acid (10g) of last gained in the Iso Butyl Acetate (250mL), it will be extracted into cold 0.1N NaOH (in 3 * 250mL).The extract that merges is washed with ether (250mL), and with 6N HCl it slowly being acidified to the pH value then is 4.(2 * 250mL) extract carboxylic acid, and Dichlorodiphenyl Acetate isopropyl ester layer carries out drying and concentrates, and obtains pure basically product, and this is used for next step with Iso Butyl Acetate.
Note: oxygenant (H 5IO 6/ CrO 3) as described in Tetrahedron Letters 39 (1998) 5323-5326, be prepared, but be to use the CH of HPLC level 3CN (containing 0.5% water); Do not add entry.
1H?NMR(CD 3COCD 3)δ8.05(2H，d)，7.95(2H，d)，7.8(2H，d)，7.65(2H，d)，4.45-4.55(1H，m)，3.55-3.6(1H，m)，3.2(3H，s)，2.8-3.0(broad?m，NH/OH)1.95-2.05(1H，m)，1.55-1.6(2H，m)，0.9-1.0(6H，m).
Step 7:N 1-(cyanogen methyl)-N 2The preparation of-{ (1S)-2,2,2-three fluoro-1-[4 '-((methylsulfonyl))-1,1 '-xenyl-4-yl] ethyl }-L-leucine amide
In DMF (200mL) solution, add benzotriazole-1-base oxygen base three (dimethylamino) phosphorus  hexafluorophosphate (11.6g), aminoacetonitriles hydrochloride (3.94g) and mixture is cooled to 0 ℃ from the acid (9g) of step 7.Drip to add triethylamine (9.9mL), mixture is warming up to room temperature and stirred 16 hours.Be poured in ice and the saturated sodium bicarbonate aqueous solution, (3 * 100mL) extract with ether.The extract that merges with the salt water washing, with dried over mgso and under vacuum except that desolvating.By chromatography at SiO 2Last use ethyl acetate and hexane (1: 1) carry out purifying to resistates.Then title compound is stirred in ether 16 hours, filter and dry (140.5 ℃ of fusing points).
1H?NMR(CD 3COCD 3)δ8.0(2H，d)，7.95(2H，d)，7.8(2H，d)，7.65(2H，d)，4.35-4.45(1H，m)，4.1-4.2(2H，m)，3.45-3.55(1H，m)，3.15(3H，s)，2.65-2.7(1H，m)，1.85-1.95(1H，m)，1.4-1.6(2H，m)，0.85-0.95(6H，m).
Embodiment 5
N 1-(1-cyano group cyclopropyl)-4-fluoro-N 2The preparation of-{ (1S)-2,2,2-three fluoro-1-[4 '-((methylsulfonyl))-1,1 '-xenyl-4-yl] ethyl }-L-leucine amide
Figure A20048002452000561
Step 1:(3S)-and the 3-[(tertbutyloxycarbonyl) amino]-the 4 hydroxybutyric acid benzyl ester
N-(tertbutyloxycarbonyl)-L-aspartic acid 4-benzyl ester (30g) is dissolved in the glycol dimethyl ether (90mL), solution is cooled to-5 ℃.Add N-methylmorpholine (10.32mL) then,, so make temperature keep below-10 ℃ succeeded by adding isobutyl chlorocarbonate (12.7mL).With mixture ageing 0.5 hour.Rapid filtration of solid and spent glycol dme (90mL) are washed.In being cooled to-50 ℃ filtrate, add sodium borohydride (4.4g) aqueous solution (45mL) carefully, add by mode like this, to maintain the temperature between-30 ℃ and-15 ℃.After adding hydride, water (500mL) is pressed certain way add, make temperature keep below-15 ℃.With suspension filtered, water (400mL) washs solid and is dry, obtains (3S)-3-[(tertbutyloxycarbonyl) amino]-the 4 hydroxybutyric acid benzyl ester.
1H?NMR(CD 3COCD 3)δ7.3-7.45(5H，m)，5.85-5.95(1H，NH)，5.15(2H，s)，3.95-4.1(2H，m)，3.5-3.7(2H，m)，2.55-2.75(2H，m)，1.4(9H，s).
Step 2:[(4S)-and 2-oxo-1,3- azoles alkane-4-yl] the acetate benzyl ester
In the alcohol that is dissolved in ethylene dichloride (925mL) (95.7g) of step 1, add pyridine (625mL) and mixture is cooled to 0-5 ℃.Anhydrous tosic acid acid anhydride (105.7g) is added, mixture is warming up to room temperature and stirred 1 hour.Being heated 90 ℃ then kept 2 hours.With the mixture cooling, (3 * 600mL) wash with methylene dichloride (1000mL) dilution with 1N HCl.With salt water washing organic layer, remove with dried over sodium sulfate with under vacuum and to desolvate.By chromatography at SiO 2Resistates is carried out purifying, and usage ratio is 1: 1 ethyl acetate and a hexane, uses ethyl acetate then, obtain [(4S)-and 2-oxo-1,3- azoles alkane-4-yl] the acetate benzyl ester.
1H?NMR(CD 3SOCD 3)δ7.8(1H，NH)，7.3-7.45(5H，m)，5.05-5.15(2H，m)，4.4-4.5(1H，m)，4.1-4.2(1H，m)，4.0-4.05(1H，m)，3.6-3.8(2H，m).
Step 3:(4S)-and 4-(2-hydroxy-2-methyl propyl group)-1,3- azoles alkane-2-ketone
Under-20 ℃, methyl-magnesium-bromide (the 3M diethyl ether solution of 227mL) is joined in the mixture of toluene (340mL) and THF (340mL).Then the hot THF solution (170mL) of the ester (40g) of step 2 is dripped and add, keep temperature to be lower than-10 ℃, placed mixture 2 hours.Mixture is slowly joined in the mixture of water (1000mL) and acetate (200mL) then, and at room temperature stirred the mixture 2 hours.Water layer separated and with organic layer further water (2 * 200mL) extract.Use methylene dichloride and continuous extractor that product is extracted from the water layer that merges.By means of heptane with dichloromethane extraction liquid evaporate to dryness.By chromatography at SiO 2Last use ethanol and methylene dichloride (1: 30) carry out purifying to resistates, obtain (4S)-4-(2-hydroxy-2-methyl propyl group)-1,3- azoles alkane-2-ketone.
1H?NMR(CD 3COCD 3)δ6.1-6.4(1H，NH)，4.45-4.55(1H，m)，4.1-4.2(1H，m)，3.95-4.05(1H，m)，3.7(1H，s)，1.65-1.85(2H，m)，1.25(6H，m).
Step 4:(4S)-and 4-(2-fluoro-2-methyl-propyl)-1,3- azoles alkane-2-ketone
Methylene dichloride (100mL) solution that derives from the alcohol (47.8g) of step 3 is joined in methylene dichloride (500mL) solution of (diethylamino) sulfur trifluoride (48.5g).Mixture is warming up to room temperature and stirred 1 hour.Carefully it is joined 0 ℃ saturated NaHCO then 3In the mixture of the aqueous solution (800mL).Organic layer is separated, use saturated NaHCO 3Solution washing.Further use methylene dichloride (100mL) aqueous layer extracted, the dichloromethane layer that merges is carried out drying and concentrated.Use ethyl acetate and hexane (1: 5), use ethyl acetate then, by chromatography at SiO 2Resistates is carried out purifying, obtain (4S)-4-(2-fluoro-2-methyl-propyl)-1,3- azoles alkane-2-ketone.
1H?NMR(CD 3SOCD 3)δ7.6(1H，NH)，4.4-4.5(1H，m)，3.95-4.05(1H，m)，3.9-3.95(1H，m)，1.8-1.95(2H，m)，1.25-1.4(6H，2s).
Step 5:(2S)-2-amino-4-fluoro-4-methylpentane-1-alcohol.
Add potassium hydroxide (21.9g) in the fluoro derivatives (21.0g) of the step 4 in being dissolved in 90% aqueous ethanolic solution (216mL).Mixture was heated 4 hours under refluxing, be cooled to room temperature.Then mixture is concentrated and (3 * 300mL) carry out coevaporation with toluene.Be dissolved in resistates in the methylene dichloride (500mL) and stirred 0.5 hour.(3 * 100mL) wash diatomite with the suspension filtration over celite with methylene dichloride.Filtrate is concentrated into drying, obtains (2S)-2-amino-4-fluoro-4-methylpent-1-alcohol.
1H?NMR(CD 3OD)δ3.4-3.5(1H，m)，3.2-3.3(1H，m)，3.0-3.1(1H，m)，1.5-1.7(2H，m)，1.35(3H，s)，1.3(3H，s).
Step 6:(2S)-and the 1-{[tertiary butyl (dimethyl) silyl] the oxygen base }-4-fluoro-4-methylpentane-2-amine
The amino alcohol (21.0g) that derives from step 5 is dissolved in the methylene dichloride (300mL), solution is cooled to 0 ℃.4-(dimethylamino) pyridine (0.051g) and tert-butyldimethylsilyl chloride thing (21g) are added, add triethylamine (25mL) then.At room temperature stir the mixture and spend the night.Reaction mixture slowly poured in 0 ℃ the saturated aqueous ammonium chloride, and (3 * 300mL) extract with methylene dichloride.Remove with salt water washing organic layer, with dried over sodium sulfate with under vacuum and to desolvate, obtain (2S)-1-{[tertiary butyl (dimetylsilyl] the oxygen base }-4-fluoro-4-methylpentane-2-amine.
1H?NMR(CD 3OD)δ3.6-3.65(1H，m)，3.4-3.5(1H，m)，3.1-3.2(1H，m)，1.6-1.8(2H，m)，1.35-1.45(6H，m)，0.93(9H，s)，0.1(6H，s).
Step 7:(2S)-and the 1-{[tertiary butyl (dimethyl) silyl] the oxygen base }-4-fluoro-4-methyl-N-[(1E)-2,2,2-trifluoro ethylidene] pentane-2-amine.
In the amine that derives from step 6 (31.5g) that is dissolved in benzene (126mL), add trifluoro acetaldehyde methyl hemiacetal (21.6mL).Heated solution spends the night under refluxing, and uses the Dean-Stark trap to collect water.Reaction mixture is cooled to room temperature and is concentrated into drying.At SiO 2Last 4% the ethyl acetate isohexane solution of using carries out purifying to resistates, obtain (2S)-1-([tertiary butyl (dimethyl) silyl] oxygen base }-4-fluoro-4-methylpentane-2-amine.
1H?NMR(CD 3COCD 3)δ7.9-7.95(1H，m)，3.75-3.85(1H，m)，3.7-3.75(1H，m)，3.53-3.6(1H，m)，1.9-2.0(2H，m)，1.3-1.4(6H，m)，0.9(9H，s)，0.1(3H，s)，0.05(3H，s).
Step 8:(2S)-2-{[(1S)-and 1-(4-bromophenyl)-2,2, the 2-trifluoroethyl] amino }-4-fluoro-4-methylpentane-1-alcohol
To-75 ℃ 1, in THF (4mL) solution of 4-dibromobenzene (0.26g), add n-BuLi (the 2.5M hexane solution of 0.42mL) and mixture placed 20 minutes.Add step 7 imines (0.329g) THF (2mL) solution and mixture placed 2 hours.Then mixture is joined water (5OmL), NH 4In the mixture of Cl (1g) and trash ice.(2 * 25mL) extract and with the ethyl acetate layer drying and the evaporate to dryness that merge it with ethyl acetate.
Repeat identical step, but be to use dibromobenzene (1.2g), n-BuLi (1.84mL) and imines (1.38g.) and as above the processing of reaction mixture.The resistates that obtains according to two kinds of preparation methods that merges is dissolved among the THF (10mL), and is cooled to 0 ℃.Add n-butyl ammonium fluoride (the 1M THF solution of 6mL) and under+5 ℃, stirred the mixture 16 hours.Be poured into then in the mixture of water (50mL), ammonium chloride (1g) and trash ice, and organic layer is separated.(2 * 15mL) aqueous layer extracted and the organic layer that is combined carry out drying and concentrate further to use ethyl acetate.At SiO 2Last use ethyl acetate and hexane (1: 5) carry out purifying to resistates, obtain (2S)-2-{[(1S)-1-(4-bromophenyl)-2,2, the 2-trifluoroethyl] amino }-4-fluoro-4-methylpent-1-alcohol.
1H(CD 3COCD 3)δ7.65(2H，m)，7.5(2H，m)，4.5-4.6(1H，m)，3.8(1H，m)，3.6(1H，m)，3.3-3.4(1H，m)，2.85-2.0(1H，m)，2.55(1H，m)，1.7-1.9(2H，s)，1.3-1.4(6H，m).
Step 9:N-[(1S)-and 1-(4-bromophenyl)-2,2, the 2-trifluoroethyl]-4-fluoro-L-leucine.
With H 5IO 6/ CrO 3Suspension (the 0.44M CH of 529mL 3CN solution; Note) is cooled to 0 ℃ and will derive from the CH of the alcohol (1.55g) of step 8 3CN (5mL) solution drips and adds.Under 0～5 ℃, stirred the mixture 3.5 hours.Under vigorous stirring, be poured into the pH value and be 4 Na 2HPO 4(3 * 50mL) extract mixtures (200mL) and with ether.With ether extraction liquid water and salt solution (1: the 1) washing that merges, use NaHSO then 3Dilute solution and salt water washing.With dried over sodium sulfate and under vacuum with the solvent evaporate to dryness, obtain N-[(1S)-1-(4-bromophenyl)-2,2, the 2-trifluoroethyl]-4-fluoro-L-leucine, in next step, use like this.
Note: oxygenant (H 5IO 6/ CrO 3) as described in Tetrahedron Letters 39 (1998) 5323-5326, be prepared, but be to use the CH of HPLC level 3CN (containing 0.5% water); Do not add entry.
Step 10:N 2-[(1S)-and 1-(4-bromophenyl)-2,2, the 2-trifluoroethyl]-N 1-(1-cyano group cyclopropyl)-4-fluoro-L-leucine amide.
Diisopropylethylamine (4.2mL) joined 0 ℃ the acid that derives from step 9 (1.5g), 1-amino-1-cyclopropane nitrile hydrochloride (1.18g), 0-(7-azepine benzo triazol-1-yl)-N, N, N ', in N '-tetramethyl-urea  hexafluorophosphate (1.94g) and dimethyl formamide (5mL) suspension, mixture at room temperature reacted 48 hours.Then it is poured in ice and the rare aqueous ammonium chloride solution.'s rare Na of 3 with ethyl acetate and ether (1: 1) extraction mixture with the organic layer that merges with the pH value 2HPO 4Solution and salt water washing.With the solvent evaporate to dryness and use ethyl acetate and hexane (1: 2) by chromatography at SiO 2Go up resistates is carried out purifying, obtain N 2-[(1S)-and 1-(4-bromophenyl)-2,2, the 2-trifluoroethyl]-N '-(1-cyano group cyclopropyl)-4-fluoro-L-leucine amide, have the abundant purity that is used for next step reaction.
1H?NMR(CD 3COCD 3)δ8.15(1H，NH)，7.6(2H，m)，7.45(2H，m)，4.35-4.45(1H，m)，3.45-3.55(1H，m)，1.9-2.1(2H，m)，1.75-1.85(1H，NH)，1.35-1.55(8H，m)，1.1-1.15(1H，m)，0.95-1.05(1H，m).
Step 11:N 1-(1-cyano group cyclopropyl)-4-fluoro-N 2-(1S)-2,2,2-three fluoro-1-[4 '-(methylthio group)-1,1 '-xenyl] ethyl }-the L-leucine amide.
Allow nitrogen gas stream pass through suspension 15 minutes, suspension is by the bromide that derives from step 10 (0.338g.), 4-(methylthio group) phenyl-boron dihydroxide (0.252g), 2M Na 2CO 3(0.8mol) and DMF (4mL) constitute.Add PdCl then 2Dppf (0.1g) and reaction is warming up to 85 ℃, and under nitrogen, stirred 5 hours.Mixture is cooled to room temperature, in ethyl acetate dilution and being poured into water (50mL) and icing.Ethyl acetate layer is separated, and further water layer is extracted with ethyl acetate.The dry acetic acid ethyl acetate extract that merges and under vacuum, remove and desolvate.Use ethyl acetate and hexane (1: 2) by chromatography at SiO 2Go up resistates is carried out purifying, obtain N 1-(1-cyano group cyclopropyl)-4-fluoro-N 2-(1S)-2,2,2-three fluoro-1-[4 '-(methylthio group)-1,1 '-xenyl-4-yl] ethyl }-the L-leucine amide.
1H?NMR(CD 3COCD 3)δ8.15(1H，NH)，7.1-7.2(4H，m)，7.5-7.55(2H，m)，7.35-7.4(2H，m)，4.3-4.4(1H，m)，3.45-3.55(1H，m)，2.75-2.8(1H，NH)，2.5(3H，s)，1.9-2.05(2H，m)，1.3-1.5(8H，m)，1.0-1.1(1H，m)，0.85-0.95(1H，m).
Step 12:N 1-(1-cyano group cyclopropyl)-4-fluoro-N 2The preparation of-{ (1S)-2,2,2-three fluoro-1-[4 '-((methylsulfonyl))-1,1 '-xenyl-4-yl] ethyl }-L-leucine amide
In the toluene (5mL) of 0 ℃ the sulfide that derives from step 11 (0.265g) and methylene dichloride (5mL) solution, add Na 2WO 42H 2O (0.002g) and n-Bu 4NHSO 4(0.01g).Slowly add 30% hydrogen peroxide (0.137mL) then and at room temperature stirred the mixture 3 hours.Then compound is poured on the mixture of ice, rare sodium thiosulfate solution and ethyl acetate.Organic layer is separated, and further water layer is extracted with ethyl acetate.With the organic layer salt water washing that merges, use dried over mgso and under vacuum, remove to desolvate to obtain resistates, at SiO 2This resistates of last purifying uses ethyl acetate, hexane and methylene dichloride (1: 1: 0.1) as elutriant.Resistates is ground in ether, obtain N 1-(1-cyano group cyclopropyl)-4-fluoro-N 2-(1S)-2,2,2-three fluoro-1-[4 '-((methylsulfonyl))-1,1 '-xenyl-4-yl] ethyl }-the L-leucine amide.
1H?NMR(CD 3COCD 3)δ8.2(1H，NH)，8.05-8.1(2H，m)，7.95-8.0(2H，m)，7.8(2H，m)，7.65(2H，m)，4.35-4.45(1H，m)，3.5-3.6(1H，m)，3.2(3H，s)，2.8-2.9(1H，NH)，1.9-2.1(2H，m)，1.3-1.5(8H，m)，1.05-1.15(1H，m)，0.9-1.0(1H，m).
Embodiment 6
N 2-[1-(3-bromophenyl)-2,2,2-trifluoroethyl]-N 1Synthesizing of-(cyanogen methyl)-L-leucine amide
Use embodiment 1 described step, will be wherein 2,2, the 2-trifluoroacetophenone is replaced by 1-(3-bromophenyl)-2,2, and 2-trifluoro ethyl ketone prepares title compound.
MS(+ESI)：406.0，408.1[M+1] +.
Embodiment 7
N 1-(cyanogen methyl)-N 2-[2,2,2-three fluoro-1-(4-piperazine-1-phenyl) ethyl]-L-leucine amide
Figure A20048002452000622
Step 1:N 2-(1-{4-[4-(tertiary butyl carboxyl) piperazine-1-yl] phenyl }-2,2, the 2-trifluoroethyl)-N 1-(cyanogen methyl)-L-leucine amide
With N 2-[1-(4-bromophenyl)-2,2,2-trifluoroethyl]- N 1-(cyanogen methyl)-L-leucine amide (embodiment 2) (100mg, 0.25mmol), three (dibenzalacetone) two palladium (2.3mg, 0.0025mmol), xenyl-2-base (di-t-butyl) phosphine (3mg, 0.01mmol), potassiumphosphate (74mg, 0.35mmol) and tertiary butyl piperazine-1-carboxylicesters (56mg, 0.3mmol) glycol dimethyl ether (0.5mL) solution be cooled to-78 ℃, high vacuum lower pumping 3 minutes, then nitrogen is imported in the flask.Then mixture was heated 1 hour down at 80 ℃.Behind the cool to room temperature, directly be applied to mixture on the silica gel adsorption column and use the ethyl acetate/hexane wash-out, obtain title compound.
Step 2:N 1-(cyanogen methyl)-N 2-[2,2,2-three fluoro-1-(4-piperazine-1-base phenyl) ethyl]-L-leucine amide
To the compound that derives from step 1 (139mg, 0.27mmol) 1, (53 μ L 0.82mmol), stir the mixture and spend the night to add methylsulfonic acid in 4-dioxane (0.5mL) solution.Add ethyl acetate, then mixture used saturated sodium bicarbonate and salt water washing, used dried over mgso, filtration and vaporising under vacuum solvent.With 93% methylene dichloride, 0.6% ammoniacal liquor and 6.4% methanol-eluted fractions, obtain title compound by the silica gel chromatography purifying.
MS(+ESI)：412.2[M+1] +.
Embodiment 8
N 2-((1S)-1-{4 '-[1-(aminocarboxyl) cyclopropyl] xenyl-4-yl }-2,2, the 2-trifluoroethyl)-N 1Synthesizing of-(1-cyano group cyclopropyl)-4-fluoro-L-leucine amide
The preparation of step 1:1-(4-bromophenyl) cyclopropane nitrile
In 22mL sodium hydroxide (50%W/W in the water) solution of the 4-bromophenyl acetonitrile (18.0g) under room temperature, add 1-bromo-2-monochloroethane (12.0mL) and benzyl trimethyl ammonium chloride (627mg).Mixture is 60 ℃ of heated overnight.Reaction mixture is cooled to room temperature, adds ether (300mL).Water (100mL), hydrogenchloride (100mL contains 10%HCl in the water) and salt water washing ether layer.Organic layer removes with dried over mgso with under vacuum and desolvates.By using ether and hexane to grind the purifying resistates, obtain title compound.
1H?NMR(CD 3COCD 3)δ7.60(2H，d)，7.35(2H，d)，1.74-1.80(2H，m)，1.52-1.57(2H，m).
The preparation of step 2:1-(4-bromophenyl) cyclopropane-carboxylic acid
Add 56mL sodium hydroxide solution (NaOH W/W in water is 25%) in ethanol (110mL) solution of (4-bromophenyl) the cyclopropane nitrile of the 1-that derives from step 1 under room temperature (13g).With mixture 100 ℃ of heated overnight.It is cooled to room temperature, pours in ice and the hydrogenchloride (1N), and (2 * 100mL) extract with methylene dichloride.The extract that merges with the salt water washing, with dried over mgso and under vacuum except that desolvating, obtain title compound.
1H?NMR(CD 3COCD 3)δ7.50(2H，d)，7.35(2H，d)，1.53-1.60(2H，m)，1.18-1.22(2H，m).
The preparation of step 3:1-(4-bromophenyl) cyclopropane carboxamide
In chloroform (60mL) solution of-15 ℃ the 1-that derive from step 2 (4-bromophenyl) cyclopropane-carboxylic acids (1.5g), slowly add isobutyl chlorocarbonate (900 μ L) and triethylamine (1.1mL).Reaction mixture was stirred 2 hours down at-15 ℃.Make it saturated and stirred 10 minutes down with ammonia then at-15 ℃.The homologation reaction mixture was at room temperature placed 1 hour, was poured into water (60mL) neutralization carrying out layering then.With methylene dichloride (2 * 60mL) aqueous layer extracted.With the extract salt water washing that merges, with dried over mgso and under vacuum except that desolvating.By using the volatilization of ether and hexane that resistates is carried out purifying, obtain title compound.
1H?NMR(CD 3COCD 3)δ7.54(2H，d)，7.40(2H，d)，6.45(1H，bs)，5.96(1H，bs)，1.42-1.48(2H，m)，0.98-1.02(2H，m).
Step 4:N 1-(1-cyano group cyclopropyl)-4-fluoro-N 2The preparation of-{ (1S)-2,2,2-three fluoro-1-tetramethyl-s-1,3,2-two oxa-boron heterocycle pentane-2-yls] ethyl }-L-leucine amide
Allow nitrogen gas stream by deriving from the N of embodiment 5 steps 9 2-[(1S)-and 1-(4-bromophenyl)-2,2, the 2-trifluoroethyl]-N 1DMF (40mL) suspension of-(1-cyano group cyclopropyl)-4-fluoro-L-leucine amide, two (tetramethylethylene glycolization) two boron (1.24g) and potassium acetate (1.53g) 15 minutes.Add the title complex (1: 1) of catalyzer [1,1 '-two (diphenyl phosphine)-ferrocene] palladium chloride (II) and methylene dichloride (181mg) then, mixture is warming up to 65 ℃, under nitrogen, spend the night.Mixture is cooled to room temperature, with ethyl acetate and hexane (1: 1,100mL) dilute and be poured on water (50mL) and ice on (50g).Organic layer is separated, and (1: 1,3 * 50mL) carried out aqueous layer extracted further to use ethyl acetate and hexane.The extraction liquid that merges with the salt water washing and use dried over mgso.Removing desolvates obtains resistates, uses ethyl acetate and hexane (1: 3～1: 2), and this resistates is passed through SiO 2Chromatogram is carried out purifying, obtains title compound.
1H?NMR(CD 3COCD 3)δ8.15(1H，bs)，7.78(2H，d)，7.50(2H，d)，4.31-4.40(1H，m)，3.47-3.54(1H，m)，2.72-2.80(2H，m)，1.32-1.48(9H，m)，1.05-1.11(1H，m)，0.87-0.95(1H，m).
Step 5:N 2-((1S)-1-{4 '-[1-(aminocarboxyl) cyclopropyl] xenyl-4-yl }-2,2, the 2-trifluoroethyl)-N 1The preparation of-(1-cyano group cyclopropyl)-4-fluoro-L-leucine amide
With nitrogen gas stream by the borate (150mg) that derives from step 4,1-(4-bromophenyl) cyclopropane carboxamide (110mg) and the 2M Na that derives from step 3 2CO 3DMF (4ml) solution of (400 μ L) 15 minutes.Add catalyzer [1,1 '-two (diphenyl phosphine)-ferrocene] palladium chloride (I) then and be warming up to 80 ℃, under nitrogen, kept 3 hours with the title complex (1: 1) of methylene dichloride (12mg) with mixture.Mixture is cooled to room temperature, pours in ice (10g) and the saturated sodium bicarbonate aqueous solution (20mL), and (3 * 30mL) extract with 50% ethyl acetate.The extraction liquid that merges with the salt water washing and use dried over mgso.Removing desolvates obtains resistates, uses ethyl acetate and hexane (50～70%) as elutriant, with this resistates by chromatography at SiO 2On carry out purifying, by using the ether volatilization to carry out purifying, obtain title compound then.
1H?NMR(CD 3COCD 3)δ8.20(1H，bs)，7.75(2H，d)，7.70(2H，d)，7.60(2H，d)，7.55(2H，d)，6.37(1H，bs)，5.87(1H，bs)，4.35-4.43(1H，m)，3.52-3.58(1H，m)，1.92-2.05(2H，m)，1.42-1.50(6H，m)，1.35-1.42(4H，m)，1.03-1.12(3H，m)，0.92-0.98(1H，m).
Embodiment 9
N 2-[(1S)-and 1-(4-bromophenyl)-2,2, the 2-trifluoroethyl]-N 1-(1-cyano group cyclopropyl)-4,4-two fluoro-L-norvaline acid amides
Figure A20048002452000651
The preparation of step 1:N-((benzyloxy) carbonyl)-3-iodo-L-alanine methyl ester
(25g in ethyl acetate 104mmol) (200mL) solution, adds the diethyl ether solution of diazomethane, until presenting lasting slight yellow to carbobenzoxy-(Cbz)-L-Serine.With the solvent vaporising under vacuum.In resistates, add N, and dinethylformamide (400mL) and methyl triple phenoxyl phosphine  iodide (50g, 110mmol).Stirred the mixture 15 minutes, and added methyl alcohol (15mL) then, pour 20% Sulfothiorine then in mixture, use ethyl acetate: hexane is that 1: 1 mixture (2L) extracts.Water and salt solution (3 *) washing organic layer, use dried over mgso, the filtration and under vacuum with solvent evaporation.Use ethyl acetate and hexane, by silica gel chromatography purifying resistates.With compound grinding in ether/hexane, the filtration and air-dry that obtains, obtain N-((benzyloxy) carbonyl)-3-iodo-L-alanine methyl ester.
The preparation of step 2:N-((benzyloxy) carbonyl)-oxo-L-norvaline methyl esters
With derive from step 1 N-((benzyloxy) carbonyl)-3-iodo-L-alanine methyl ester (10g, 27.5mmol), the zinc in the benzene (110mL)-copper idol (3.3g) and N,N-dimethylacetamide (7.4mL) supersound process 2 hours in ultra sonic bath.After during this, add 3 batches of glycol dibromides (0.24mL) and trimethylchlorosilane (0.17mL).In this mixture, add then two (triphenylphosphine) Palladous chlorides (0.958g, 1.4mmol) and Acetyl Chloride 98Min. (2.5mL 35.2mmol), heats mixture 2 hours at 70 ℃.After being cooled to room temperature, use ethyl acetate filtering mixt on diatomite, use saturated ammonium chloride solution and salt solution (2 *) washing organic layer then, use dried over mgso, filtration and vaporising under vacuum solvent.Use ethyl acetate and hexane that resistates is carried out purifying by silica gel chromatography, obtain N-((benzyloxy) carbonyl)-4-oxygen-L-norvaline methyl esters.
Step 3:N-((benzyloxy) carbonyl)-4, the preparation of 4-two fluoro-L-norvaline methyl esters
(1.3g slowly adds DAST (2.46mL) in methylene dichloride 4.65mmol) (20mL) and methyl alcohol (0.019mL) solution to N-under 0 ℃ ((benzyloxy) carbonyl)-4-oxygen-L-norvaline methyl esters.Remove ice bath and use hot water bath (57 ℃) to replace.Replace hot water bath 3 times, mixture is at room temperature stirred spend the night then.Mixture slowly is poured over cold saturated NaHCO 3On, use ethyl acetate extraction, use the salt water washing, use dried over mgso, filter and under vacuum with solvent evaporation.By silica gel chromatography, use ethyl acetate and hexane purifying resistates, obtain N-((benzyl two is fluorine-based) carbonyl)-4,4-two fluoro-L-norvaline methyl esters.
Step 4:(1S)-3, the preparation of 3-two fluoro-1-(methylol) butyl carbamic acid benzyl base esters
To N-((benzyloxy) carbonyl)-4, (1.59g adds lithium chloride (919mg) in ethanol 5.29mmol) (50mL) solution to 4-two fluoro-L-norvaline methyl esters, stirs the mixture 10 minutes.Slowly add sodium borohydride (820mg), stirred the mixture 2 hours.Then, add another part sodium borohydride (100mg), continue to stir 30 minutes.Water (20mL) diluted mixture thing and neutralize at leisure with 1N HCl is succeeded by adding another water gaging such as grade.With ethyl acetate (2 *) extraction mixture, with the salt water washing, with dried over mgso, filtration and under vacuum with solvent evaporation, obtain (1S)-3,3-two fluoro-1-(methylol) butyl carbamic acid benzyl base esters.
Step 5:(2S)-and 1-((tertiary butyl (dimethyl) silyl) oxygen base)-4, the preparation of 4-difluoro penta-2-amine
To (1S)-3,3-two fluoro-1-(methylol) butyl carbamic acid benzyl base esters (derive from ethanol (25mL) solution of step 4), be added in palladium on the gac (10%, 150mg), at H 2(H under the atmosphere 2Bubble) 2h that stirs the mixture.Add methylene dichloride, filtering mixt on diatomite.The vaporising under vacuum solvent.Resistates is dissolved in the methylene dichloride (15mL), and with triethylamine (1mL), N, N-dimethyl aminopyridine (10mg) and tertiary butyl dimethyl chloride add for silicomethane (844mg).Stir the mixture and spend the night, add entry and salt solution then.With ethyl acetate (2 *) extraction mixture, with the salt water washing, with dried over mgso, filtration and under vacuum with solvent evaporation, obtain (2S)-1-((tertiary butyl (dimethyl) silyl) oxygen base)-4,4-two amyl fluorides-2-amine.
Step 6:(2S)-and 1-((tertiary butyl (dimethyl) silyl) oxygen base)-4, the preparation of 4-two fluoro-N-((1E)-2,2,2-trifluoro ethylidene) pentane-2-amine
To derive from (the 2S)-1-((tertiary butyl (dimethyl) silyl) oxygen base)-4 of step 5, (80%, benzene 0.9mL) (20mL) solution refluxes with the Dean-Stark instrument and spends the night for 4-two amyl fluorides-2-amine and trifluoro acetaldehyde methyl hemiacetal.With the solvent vaporising under vacuum, use ethyl acetate and hexane to carry out the purifying resistates by silica gel chromatography, obtain (2S)-1-((tertiary butyl (dimethyl) silyl) oxygen base)-4,4-two fluoro-N-(1E)-2,2,2-trifluoro ethylidene) pentane-2-amine.
Step 7:(2S)-and 2-((1S)-1-(4-bromophenyl)-2,2,2-trifluoroethyl) amino)-4, the preparation of 4-two amyl fluorides-2-amine
To-78 ℃ 1, in THF (5.2mL) solution of 4-dibromobenzene (330 milligrams), add the hexane solution (0.52mL) of the n-BuLi of 2.5M, with aging 30 minutes of solution.Then, add (2S)-1-((tertiary butyl (dimethyl) silyl) oxygen base)-4, THF (5.2mL) solution of 4-two fluoro-N-((1E)-2,2,2-trifluoro ethylidene) pentane-2-amine (333mg).Mixture was stirred 45 minutes down at-78 ℃, is poured over then in the cold saturated ammonium chloride, with ethyl acetate (2 *) extraction, with the salt water washing, with dried over mgso, filtration and under vacuum with solvent evaporation.Resistates is dissolved among the ice-water bath refrigerative THF (10mL), and adding tetra-n-butyl Neutral ammonium fluoride (1M in THF, 1.5mL).The 1h that stirs the mixture under 0 ℃ pours in the cold water, with ethyl acetate (2 *) extraction, uses the salt water washing, use dried over mgso, filter and under vacuum with solvent evaporation, obtain (2S)-2-(((1S)-1-(4-bromophenyl)-2,2, the 2-trifluoroethyl) amino)-4,4-difluoro penta-1-alcohol.
Step 8:N 2-[(1S)-and 1-(4-bromophenyl)-2,2, the 2-trifluoroethyl]-N 1-(1-cyano group cyclopropyl)-4, the preparation of 4-two fluoro-L-norvaline acid amides
With H 5IO 6/ CrO 3Suspension (the 0.44M CH of 27mL 3CN solution; Note) is cooled to 0 ℃, drips the CH that adds the alcohol (740mg) that derives from step 7 3CN (10mL) solution.Mixture was stirred 4 hours down at 0 ℃, add H in addition again 5IO 6/ CrO 3(the 0.44MCH of 2 * 10mL 3CN solution).Under vigorous stirring, mixture poured into the pH value then and is 4 Na 2HPO 4In the buffered soln, use the ethyl acetate extraction mixture,, use rare NaHSO then with salt solution (2 *) washing 3The aqueous solution and salt water washing.Use the dried over mgso mixture, filter and, obtain 680mg N-[(1S the solvent evaporate to dryness)-1-(4-bromophenyl)-2,2, the 2-trifluoroethyl]-4,4-two fluoro-L-norvalines, with its like this sample be used for next step.
Note: oxygenant (H 5IO 6/ CrO 3) as described in Tetrahedron Letters 39 (1998) 5323-5326, be prepared, but be to use the CH of HPLC level 3CN (containing 0.5% water); Do not add entry.
Triethylamine (0.42mL) is joined in the mixture that derives from above acid (340mg), 1-amino-1-cyclopropane nitrile hydrochloride (227mg), benzotriazole-1-base-oxygen base tripyrrole alkyl phosphorus  hexafluorophosphate (498mg) and dimethyl formamide (4.5mL), allow mixture at room temperature react 48 hours.Be poured into then in rare sodium bicarbonate.With ether (3 *) extraction mixture, the organic layer that merges is washed with salt solution (3 *), and also filter with dried over mgso.With the solvent evaporate to dryness, use 40% ethyl acetate and hexane by chromatography purifying resistates on silica gel, obtain N 2-[(1S)-and 1-(4-bromophenyl)-2,2, the 2-trifluoroethyl]- N 1-(1-cyano group cyclopropyl)-4,4-two fluoro-L-norvaline acid amides.
MS(+ESI)：454.1，456.2[M+1] +.
Embodiment 10
N 2-[(1S)-and 1-(4-bromophenyl)-2,2, the 2-trifluoroethyl]-N 1-(cyanogen methyl)-4,4-two fluoro-L-norvaline acid amides
Use embodiment 9 described steps, the amino of the 1-in the step 8-1-cyclopropane nitrile hydrochloride is replaced with the aminoacetonitriles hydrochloride, obtains title compound.
MS(+ESI)：428，430.1[M+1] +.
Embodiment 11
4-fluoro-N 1-[(2R, 3S)-2-methyl-4-oxygen base tetrahydrofuran (THF)-3-yl]-N 2-(1S)-2,2,2-three fluoro-1-[4 '-((methylsulfonyl))-1,1 '-xenyl-4-yl] ethyl }-the L-leucine amide
Step 1:N 2-[(1S)-and 1-(4-bromophenyl)-2,2, the 2-trifluoroethyl]-4-fluoro-N 1-[(2R, 3S)-2-methyl-4-oxygen base tetrahydrofuran (THF)-3-yl]-the L-leucine amide
Triethylamine (0.63mL) is joined acid (500mg), (4S that derives from embodiment 5 steps 9,5R)-4-amino-5-methyl dihydrofuran-3 (2H)-keto hydrochloride (227mg) is (WO00/69855), in the mixture of benzotriazole-1-base-oxygen base tripyrrole alkyl phosphorus  hexafluorophosphate (744mg) and dimethyl formamide (7mL), allow mixture at room temperature react 3 hours.Be poured into then in rare sodium bicarbonate.With ether (3 *) extraction mixture, wash the organic layer that merges with salt solution (3 *), and also filter with dried over mgso.With the solvent evaporate to dryness, by chromatography at SiO 2Last use 30% ethyl acetate and hexane carry out the purifying resistates, obtain title compound.
Step 2:4-fluoro-N 1-[(2R, 3S)-2-methyl-4-oxygen base tetrahydrofuran (THF)-3-yl]-N 2-(1S)-2,2,2-three fluoro-1-[4 '-(methylthio group) xenyl-4-yl] ethyl }-the L-leucine amide
Bromide (200mg), 4-(methylthio group) phenyl-boron dihydroxide (104mg), the 2M Na of step 1 will be derived from 2CO 3The mixture of the aqueous solution (0.51mL) and DMF (3mL) stirred 15 minutes.With PdCl 2Dppf 2(17mg) be cooled to-78 ℃,, then nitrogen fed flask, under nitrogen, under 80 ℃, heat and stirred the mixture 3 hours high vacuum lower pumping 5 minutes.Mixture is cooled to room temperature,,, use dried over mgso with saturated ammonium chloride and salt solution (3 *) washing with ethyl acetate dilution, filtration and under vacuum with solvent evaporation.Use 35% ethyl acetate/hexane as elutriant, carry out purifying, obtain title compound by silica gel chromatography.
Step 3:4-fluoro-N 1-[(2R, 3S)-2-methyl-4-oxygen base tetrahydrofuran (THF)-3-yl]-N 2-(1S)-2,2,2-three fluoro-1-[4 '-((methylsulfonyl)) xenyl-4-yl] ethyl }-the L-leucine amide
In ethyl acetate (2mL) solution of the sulfide that derives from step 2 (120mg), add Na 2WO 42H 2O (1mg) and n-Bu 4NHSO 4(4mg).Slowly add 30% hydrogen peroxide (0.06mL) then, at room temperature stirred the mixture 2 hours.Further add hydrogen peroxide (0.06mL) then.Then ethyl acetate is joined in the mixture, use dense Na 2S 2O 3(2 *) and this mixture of salt water washing are used dried over mgso, filter and under vacuum with solvent evaporation.Use 55% ethyl acetate/hexane as elutriant, carry out purifying, obtain title compound by silica gel chromatography.
MS(+ESI)：559.1[M+1] +.
Embodiment 12
N 1-(1-cyano group-1-methylethyl)-N 2-{ (1S)-2,2,2-three fluoro-1-[4 '-((methylsulfonyl)) xenyl-4-yl] ethyl }-L-leucine amide synthetic
Figure A20048002452000711
The preparation of step 1:2-amino-2-methyl propane nitrile hydrochloride
Ether (50mL) solution that in 0 ℃ ammonium chloride (15.5g) water (50mL) solution, adds acetone (17mL).Slowly add water (35mL) solution of sodium cyanide (11.9g) then with a certain speed, make temperature never above 10 ℃.After adding cyanide solution, reaction mixture was stirred 1 hour, then its placement is spent the night.Ether layer is separated, with ether (2 * 30mL) aqueous layer extracted.Organic layer with the salt water washing merges obtains resistates with dried over mgso with under vacuum except that desolvating, and this resistates is diluted with methyl alcohol (80mL).At-78 ℃ of following these solution of cooling and with nitrogen saturated (use has the Ace penstock of plug valve and thermowell).The homologation reaction mixture was at room temperature placed two days.Evict from excess ammonia by air-flow, remove methyl alcohol by evaporation.Resistates is dissolved in ether (50mL) and is cooled to 0 ℃, add 40mL hydrogenchloride (in ether, being 1.0M) solution then.Stirred the mixture 30 minutes and filter, obtain title compound.
1H?NMR(CD 3SOCD 3)δ9.45(1H，s)，1.70(6H，s).
Step 2:N 1-(1-cyano group-1-methylethyl)-N 2The preparation of-{ (1S)-2,2,2-three fluoro-1-[4 '-((methylsulfonyl)) xenyl-4-yl] ethyl }-L-leucine amide
In the DMF of the acid that derives from embodiment 4 steps 6 (1.2g) (30mL) solution, add benzotriazole-1-base oxygen base three (dimethylamino) phosphorus  hexafluorophosphate (1.55g), derive from the 2-amino-2-methyl propane nitrile hydrochloride (720mg) of step 1, mixture is cooled to 0 ℃.Triethylamine (1.3mL) drip added and mixture is warming up to room temperature and stirred 72 hours.Be poured in ice and the saturated sodium bicarbonate aqueous solution, with ether (3 * 50mL) extractions.The extract that merges with the salt water washing, with dried over mgso and under vacuum except that desolvating.By chromatography at SiO 2On carry out the purifying resistates, use gradient ethyl acetate and hexane (1: 2～1: 1) as elutriant, use ether and hexane to grind then, obtain title compound.
1H?NMR(CD 3COCD 3)δ8.08(2H，d)，7.95(2H，d)，7.80(2H，d)，7.68(1H，bs)，7.65(2H，d)，4.32-4.42(1H，m)，3.43-3.52(1H，m)，3.20(3H，s)，2.65-2.75(1H，m)，1.90-2.00(1H，m)，1.57(3H，s)，1.52(3H，s)，1.52-1.57(1H，m)，1.40-1.50(1H，m)，0.90-0.98(6H，m).
Embodiment 13
N 1-(cyanogen methyl)-3-(1-methyl cyclopropyl)-N 2-2,2,2-three fluoro-1-[4 '-((methylsulfonyl))-1,1 '-xenyl-4-yl] ethyl }-ala amide
Figure A20048002452000721
Step 1:N-(diphenylmethylene)-4-methylene radical norvaline methyl esters
Under 0 ℃, (12.0g adds 1M potassium tert.-butoxide THF (49mL, 49mmol) solution in THF 47.4mmol) (118mL) solution to N-(diphenylmethylene) methyl aminoacetate.Mixture becomes bright yellow, mixture is further stirred～15min.(5.2mL 51.3mmol), at room temperature stirred the mixture 2 days to add 3-bromo-2-methacrylic.After the water quencher, extract mixture with EtOAc.Also use hexane: EtOAc (6: 1) to carry out wash-out at the enterprising circumstances in which people get things ready for a trip spectrometry of silica gel, form 2.2g as dialkyl group product than low-polarity constituents.Further wash-out is produced as the title compound of high polarity component.
1H NMR (the δ 7.62-7.18 of acetone-d6) (m, 10H), 4.72 (s, 1H), 4.64 (s, 1H), 4.20 (dd, 1H), 3.64 (s, 3H), 2.62 (dd, 1H), 2.50 (dd, 1H), 1.48 (s, 3H).
Step 2:N-[(benzyloxy) carbonyl]-4-methylene radical norvaline methyl esters
With derive from step 1 N-(diphenylmethylene)-4-methylene radical norvaline methyl esters (6.2g, 20.2mmol) and the mixture of the 0.5M HCl aqueous solution (60mL) at room temperature stir and spend the night.With all mixture Et 2O (2 *) washs.After being cooled to 0 ℃, (40mL 40mmol) adds, then with EtOAc (50mL) and benzyl chloroformate (4mL, 28mmol) adding with the 1M NaOH aqueous solution.Mixture is stirred 2h down at 0 ℃.Then the EtOAc layer is separated water (2 *) washing, dry (MgSO 4) and concentrate.In the enterprising circumstances in which people get things ready for a trip of silica gel spectrum with use hexane: EtOAc (6: 1), (3: 1) carry out wash-out then, and generation is as the title compound of water white oil.
1H NMR (the δ 7.40-7.25 of acetone-d6) (m, 5H), 6.54 (d, 1H), 5.06 (s, 2H), 4.82 (s, 1H), 4.78 (s, 1H), 4.40 (m, 1H), 3.68 (s, 3H), 2.52 (dd, 1H), 2.40 (dd, 1H), 1.74 (s, 3H).
Step 3:N-[(benzyloxy) carbonyl]-3-(1-methyl cyclopropyl)-L-alanine methyl ester
CH to 0 ℃ 2CL 2(2mL 19.5mmol), drips adding trifluoroacetic acid (1.5mL, CH 19.5mmol) then to add zinc ethyl (40mL) 2CL 2(8mL) solution.Stir after 15 minutes, with methylene iodide (1.6mL, CH 20.0mmol) 2CL 2(8mL) solution adds.Stirred the mixture 15 minutes, and produced clear solution.To derive from the N-[(benzyloxy of step 2) carbonyl]-(2.75g 9.9mmol) adds 4-methylene radical norvaline methyl esters, mixture is at room temperature stirred spend the night.After the 0.1M HCl aqueous solution (50mL) quencher, with CH 2Cl 2Layer separates, with the weak brine washing, and dry (MgSO 4) and concentrate, produce the title compound crude product.
1H NMR (δ 7.35 of acetone-d6) (m, 5H), 6.58 (d, 1H), 5.08 (m, 2H), 4.40 (m, 1H), 3.68 (s, 3H), 1.75 (dd, 1H), 1.62 (dd, 1H), 1.08 (s, 3H), 0.45 (m, 1H), 0.22 (m, 3H).
Step 4:2-hydroxyl-1-[(1-methyl cyclopropyl) methyl] the ethyl carbamic acid benzyl ester
In EtOH (40mL) that derives from the thick ester of step 3 at 0 ℃ of following refrigerative and THF (40mL) solution, add LiCl (1.7g), add NaBH then 4(1.6g).At room temperature stir the mixture and spend the night, extract with 0.5M HCl aqueous solution quencher with EtOAc.The EtOAc extraction liquid is washed dry (MgSO with weak brine (2 *) 4) and concentrate.Chromatogram is purified and is produced the title compound of 2g water white oil.
1H NMR (δ 7.35 of acetone-d6) (m, 5H), 5.98 (d, 1H), 5.06 (m, 2H), 3.85 (m, 1H), 3.72 (t, 1H), 3.50 (m, 2H), 1.60 (dd, 1H), 1.34 (dd, 1H), 1.05 (s, 3H), 0.40-0.15 (m, 4H).
Step 5:2-amino-3-(1-methyl cyclopropyl) third-1-alcohol
With 2-hydroxyl-1-[(1-methyl cyclopropyl) methyl]-the ethyl carbamic acid benzyl ester (2.0g, 7.6mmol) and 10%Pd/C (200mg) in EtOH (80mL) mixture and 2mL 1M HCl solution at H 2(bubbling) stirs and spends the night under the atmosphere.Catalyzer is leached and filtrate is concentrated, produce title compound.
1H NMR (methyl alcohol-d4) 83.72 (dd, 1H), 3.40 (dd, 1H), 3.22 (m, 1H), 1.50-1.30 (m, 2H), 1.06 (s, 3H), 0.45-0.25 (m, 4H).
Step 6:N 1-(cyanogen methyl)-3-(1-methyl cyclopropyl)-N 2-2,2,2-three fluoro-1-[4 '-((methylsulfonyl))-1,1 '-xenyl-4-yl] ethyl }-ala amide
Title compound is prepared by the amino alcohol that derives from step 5, uses and identical method described in embodiment 5 steps 6～12.
1H NMR (δ 8.06 of acetone-d6) (br s, 1H), 8.00 (d, 2H), 7.94 (d, 2H), 7.78 (d, 2H), 7.62 (d, 2H), 4.48 (m, 1H), 4.12 (m, 2H), 3.55 (m, 1H), 3.16 (s, 3H), 1.64 (m, 2H), 1.10 (s, 3H), 0.48-0.20 (m, 4H).
MS(+ESI)：494(MH +).
Embodiment 14
N 1-(cyanogen methyl)-N 2-(2,2,2-three fluoro-1-{4-[(4-methylpiperazine-1-yls) carbonyl] phenyl } ethyl)-L-leucine amide synthetic
Figure A20048002452000741
Step 1:N 2-1-[4-(hydroxycarbonyl group) phenyl] and-2,2, the 2-trifluoroethyl }-N 1-(cyanogen methyl)-L-leucine amide
Will two (triphenylphosphine) palladiums (II) of the dichloro in tributylamine (2.5mL) and the water (0.6mL) (58mg, 0.08mmol), triphenylphosphine (155mg, 0.59mmol) and N 2-[1-(4-bromophenyl)-2,2,2-trifluoroethyl]-N 1-(cyanogen methyl)-L-leucine amide (embodiment 2,1.2g, and mixture 3.0mmol) places the steel bomb that has the polytetrafluorethylecoatings coatings bar magnet.System is washed three times with carbon monoxide (each 100psi), be full of this kind gas of 300psi pressure at last.Under lasting the stirring, reaction mixture was heated 20 hours at 160 ℃.Make system cools to room temperature then, pressure is removed, the resistates that produces is distributed between EtOAc and water, adding aqueous hydrochloric acid is 2.5～3.0 to regulate the pH value.Use Na 2SO 4Dry organic layer, filtration and concentrated.Raw product is carried out purifying through chromatography, use EtOAc, hexane and acetate, produce the title compound of yellowish-orange sponge shape as elutriant.
Step 2:N 1-(cyanogen methyl)-N 2-(2,2,2-three fluoro-1-{4-[(4-methylpiperazine-1-yls) carbonyl] phenyl } ethyl)-the L-leucine amide
The N that derives from step 1 in DMF (8mL) 2-1-[4-(hydroxycarbonyl group) phenyl] and-2,2, the 2-trifluoroethyl }-N 1-(cyanogen methyl)-L-leucine amide (480mg, 1.3mmol) and benzotriazole-(1.35g is 2.6mmol) in the solution for 1-base oxygen base tripyrrole alkyl phosphorus  hexafluoro phosphonate, slowly add N methyl piperazine (0.29mL, 2.6mmol), add then triethylamine (0.54mL, 3.9mmol).Reaction mixture was at room temperature stirred 3 hours.The mixture that produces is distributed between EtOAc and water, and adding aqueous hydrochloric acid is 2.5～3.0 to regulate the pH value.With organic layer Na 2SO 4Dry, filtration and concentrated.Raw product uses MeOH, EtOAc and dense NH through chromatography purification 4OH produces the title compound of white sponge shape as elutriant.
MS(+ESI)：454.3[M+1] +.
Embodiment 15
N 1-(1S)-1-[2-(methylthio group) ethyl]-the 2-oxopropyl }-N 2-{ (1S)-2,2,2-three fluoro-1-[4 '-((methylsulfonyl)) xenyl-4-yl] ethyl }-L-leucine amide synthetic
Step 1:N 2-(tertbutyloxycarbonyl)-N 1-methoxyl group-N 1-methyl-L-methionine amide
N-tertbutyloxycarbonyl in DMF (15mL)-L-methionine(Met) (1.0g, 4.0mmol), O-(7-azepine benzo triazol-1-yl)-N, N, N ', and N '-tetramethyl-urea  hexafluorophosphate (3.3g, 8.7mmol) and N, O-dimethyl hydroxylamine hydrochloride (1.0g, 10.2mmol) ice-cold solution in, drip to add triethylamine (2.2mL, 15.8mmol).The mixture that produces was at room temperature stirred 18 hours, then at EtOAc and semi-saturation NaHCO 3Distribute between the aqueous solution.With organic layer Na 2SO 4Dry, filtration and concentrated.Raw product carries out purifying through chromatography, uses EtOAc and hexane as elutriant, produces the title compound of colourless pulpous state.
Step 2:{ (1S)-1-[2-(methylthio group) ethyl]-the 2-oxopropyl } the carboxylamine tertiary butyl ester
To the N that derives from step 1 that is cooled to-78 ℃ 2-(tertbutyloxycarbonyl)-N 1-methoxyl group-N 1-methyl-L-methionine(Met) (200mg, and the hexane solution of adding 1.4M lithium methide in THF solution 0.68mmol) (1.1mL, 1.5mmol).Stirring reaction is 2 hours under this temperature, uses the 25%W/v ammonium acetate solution with its cold quencher then.Extract mixture with EtOAc, use Na 2SO 4Dry organic layer, filtration and concentrated.Raw product carries out purifying through chromatography, uses EtOAc and hexane as elutriant, produces colourless gelationus title compound.
Step 3:(3S)-3-amino-5-(methylthio group) penta-2-keto hydrochloride
To derive from step 2 (1S)-1-[2-(methylthio group) ethyl]-the 2-oxopropyl carboxylamine tertiary butyl ester (140mg, add in 0.57mmol) 4.0M 1, the hydrogen chloride solution in the 4-dioxane (3mL, 12mmol).The gained mixture was at room temperature stirred 1 hour.Remove in a vacuum and desolvate and gained resistates and toluene (2 * 10mL) component distillations, the title compound of generation white solid.
Step 4:N 1-(1S)-1-[2-(methylthio group) ethyl]-the 2-oxopropyl }-N 2-(1S)-2,2,2-three fluoro-1-[4 '-((methylsulfonyl)) xenyl-4-yl] ethyl }-the L-leucine amide
(3S)-3-amino-5-(methylthio group) penta-2-keto hydrochloride (104mg of step 3 will be derived from; 0.57mmol), N-{ (1S)-2; 2; 2-three fluoro-1-[4 '-((methylsulfonyl))-1; 1 '-xenyl-4-yl] ethyl }-L-leucine (embodiment 4 steps 6; 127mg; 0.2mmol) and 0-(7-azepine benzo triazol-1-yl)-N; N; N ', and N '-tetramethyl-urea hexafluoro phosphorus  hydrochlorate (187mg, DMF 0.49mmol) (1mL) suspension is cooled to 10 ℃; (115 μ L 0.83mmol) slowly add with triethylamine then.Reaction mixture was at room temperature stirred 3 hours.With the mixture that produces at EtOAc and semi-saturation NaHCO 3Distribute between the aqueous solution.Use Na 2SO 4Dry organic layer, filtration and concentrated.Raw product carries out purifying through chromatography, uses EtOAc and hexane as elutriant, produces the title compound of white sponge shape.
MS(+ESI)：573.5[M+1] +.
Embodiment 16
N 1-{ (1S)-2,2,2-three fluoro-1-[4 '-((methylsulfonyl)) xenyl-4-yl] ethyl }-L-leucyl-L-methionine amide synthetic
Use the described method of embodiment 15 steps 4, will be wherein (3S)-3-amino-5-(methylthio group) penta-2-keto hydrochloride replace with L-methionine amide hydrochloride, prepare title compound, and from EtOAc and hexane (1: 2), carry out crystallization, obtain the title product of white solid.
MS(+ESI)：574.3[M+1] +.
Embodiment 17
N-{ (1S)-2,2,2-three fluoro-1-[4 '-((methylsulfonyl)) xenyl-4-yl] ethyl }-L-leucyl-L-methionine(Met) synthetic
Figure A20048002452000772
Step 1:N-{ (1S)-2,2,2-three fluoro-1-[4 '-((methylsulfonyl)) xenyl-4-yl] ethyl }-L-leucyl-L-methyl methionine synthetic
Use the described method of embodiment 15 steps 4, wherein (3S)-3-amino-5-(methylthio group) penta-2-keto hydrochloride is replaced with methyl L-methionate hydrochloride, prepare title compound, and from EtOAc and hexane (1: 2), carry out crystallization, obtain the title compound of white solid.
MS(+ESI)：589.3[M+1] +.
Step 2:N-{ (1S)-2,2,2-three fluoro-1-[4 '-((methylsulfonyl)) xenyl-4-yl] ethyl }-L-leucyl-L-methionine(Met)
To the N-{ that derives from step 1 (1S)-2; 2; 2-three fluoro-1-[4 '-((methylsulfonyl)) xenyl-4-yl] ethyl }-L-leucyl-L-methyl methionine (100mg; 0.17mmol) THF (2mL) and the ice-cold solution of methyl alcohol (0.5mL) in; drip and add 1.0N LiOH (0.25mL; 0.25mmol), at room temperature stir the solution 18 hours of gained.Solution distributes between EtOAc and water, adds 1N HCl (1mL).Use Na 2SO 4Dry organic layer, filtration and concentrated.Raw product uses EtOH, EtOAc and AcOH as elutriant through chromatography purification, produces the title compound of white sponge shape, and this compound is carried out crystallization from EtOAc and hexane (6mL, 1: 2), obtains white solid.
MS(+ESI)：575.0[M+1] +.
Embodiment 18
N 1-[(1S)-1-cyano group-3-(methylthio group) propyl group]-N 2-{ (1S)-2,2,2-three fluoro-1-[4 '-((methylsulfonyl)) xenyl-4-yl] ethyl }-L-leucine amide synthetic
The N of embodiment 16 will be derived from 1-{ (1S)-2,2,2-three fluoro-1-[4 '-((methylsulfonyl)) xenyl-4-yl] ethyl }-L-leucyl-L-methionine(Met) (350mg, 0.6mmol) and pyridine (85 μ L, 1.05mmol) 1, the 4-dioxane solution is cooled to 10 ℃.Then, (65 μ L 0.46mmol) and with reaction mixture at room temperature stirred 1 hour trifluoroacetic anhydride (TFAA) to be dripped adding.Reaction mixture obtains distributing between EtOAc and water.Use Na 2SO 4Dry organic layer, filtration and concentrated.Raw product carries out purifying through chromatography, uses EtOAc and hexane as elutriant, produces colourless gelationus title compound.
MS(+ESI)：556.3[M+1] +.
Embodiment 19
N-{ (1S)-2,2,2-three fluoro-1-[4 '-((methylsulfonyl)) xenyl-4-yl] ethyl }-L-leucyl-N 1Synthesizing of-methyl-L-methionine(Met)
Figure A20048002452000791
With the N-{ that derives from embodiment 17 (1S)-2 among the DMF; 2; 2-three fluoro-1-[4 '-((methylsulfonyl)) xenyl-4-yl] ethyl-L-leucyl-L-methionine(Met) (100mg, 0.17mmol), methylamine hydrochloride (35mg, 0.52mmol) and O-(7-azepine benzo triazol-1-yl)-N; N; N ', and N '-tetramethyl-urea  hexafluorophosphate (150mg, 0.39mmol) suspension is cooled to 10 ℃; and (110 μ L 0.79mmol) slowly add with triethylamine then.At room temperature stirred reaction mixture is 18 hours.With the mixture that produces at EtOAc and semi-saturation NaHCO 3Distribute between the aqueous solution.Use Na 2SO 4Dry organic layer, filtration and concentrated.Raw product carries out purifying through chromatography, uses EtOAc and hexane as elutriant, produces the title compound of white sponge shape, with the crystallization and produce white solid from EtOAc and hexane of this compound.
MS(+ESI)：588.1[M+1] +.
Embodiment 20
(2S)-2-{[(1S)-and 1-(4-bromophenyl)-2,2, the 2-trifluoroethyl] amino }-4,4-two chloro-N-(1-cyano group cyclopropyl) butyramide synthetic
Figure A20048002452000801
Step 1:(2S)-and 2-amino-4,4-dichloro-butyric acid ethyl ester
In the ice-cold ethanol of 25mL, and dropping adding Acetyl Chloride 98Min. (2.0mL, 28mmol).Then with (S)-2-amino-4, the 4-dichloro-butyric acid (1.0g, and 5.8mmol) [according to Chem.-Ztg, 114,249-251 (1990) and Synthesis, 1996,1419 prepare] disposable adding.With the mixture 18h that refluxes, concentrate and with resistates at saturated NaHCO 3Distribute between solution and the methylene dichloride.With separating organic matters drying (Na 2SO 4), filter and concentrate, produce the oily resistates, this resistates is carried out purifying by silicagel column, with the hexane solution wash-out of 30% ethyl acetate, produce pure title compound.
Step 2:(2S)-and 2-{ (1S)-1-(4-bromophenyl)-2,2, the 2-trifluoroethyl] amino }-4,4-dichloro-butyric acid ethyl ester
With (2S)-2-amino-4,4-dichloro-butyric acid ethyl ester (298mg, 1.49mmol), trifluoracetic acid (1S)-1-(4-bromophenyl)-2,2, (862mg is 2.2mmol) [according to J.Am.Chem.Soc. for 2-trifluoroethyl ester, 1983,105,2343-2350 prepares] and diisopropylethylamine (284mg is 2.2mmol) at 60 ℃ and N 2Even heating is 6 hours under the atmosphere.With mixture at NaHCO 3Distribute between solution and the ethyl acetate.With organic layer separation, dry (Na 2SO 4), filter and concentrate.Carrying out purifying by ISCO column chromatography (the ethyl acetate/hexane gradient is 2%～15%) provides title compound, is 87: 13 non-corresponding isomer mixture.
MS (+APCI): 438.8[M+1] and 440.8[M+3].
Step 3:(2S)-and 2-{ (1S)-1-(4-bromophenyl)-2,2, the 2-trifluoroethyl] amino }-4, the 4-dichloro-butyric acid
To (2S)-2-{[(1S)-1-(4-bromophenyl)-2,2,2-trifluoroethyl] amino }-4,4-dichloro-butyric acid ethyl ester (325mg, and adding trimethyl silicane potassium alcoholate in THF 0.74mmol) (5mL) solution (178mg, 1.39mmol).Mixture was at room temperature stirred 1.5 hours, concentrate then.Resistates is distributed between ethyl acetate and 1N HCl.With organic layer separation, dry (Na 2SO 4), filter and concentrate, obtain the buttery title compound, with this compound like this sample be used for next step.
MS(-APCI)：407.9[M-1].
Step 4:(2S)-2-{[(1S)-and 1-(4-bromophenyl)-2,2, the 2-trifluoroethyl] amino }-4,4-two chloro-N-(1-cyano group cyclopropyl) butyramide
With (2S)-2-{[(1S)-1-(4-bromophenyl)-2,2, the 2-trifluoroethyl] amino }-4,4-dichloro-butyric acid (275mg, 0.67mmol), (159mg, 1.34mmol) (305mg, mixture 0.8mmol) are dissolved among the DMF (4mL) 1-amino-1 cyclopropane nitrile hydrochloride with the HATU coupler.(0.3mL 2.1mmol) adds, and stirs the mixture and spends the night, and is poured into NaHCO then with triethylamine 3In solution and the ethyl acetate.Organic layer is separated, with salt solution, 1N HCl and salt water washing.With organic layer separation, dry (Na 2SO 4), filter and concentrate, produce 393mg oil, this oil is through the column chromatography purifying, with 6: 3: 1 toluene: ethyl acetate: methylene dichloride wash-out.Obtain the title compound of 85: 15 non-enantiomer mixtures of conduct of white solid with ether volatilization pure products.
MS(-APCI)：471.9[M-1].
1H NMR (500MHz, DMSO-d6) " main isomer ", δ 8.95 (s, 1H), 7.61 (d, 2H, 7.38 (d, 2H), 6.19 (dd, 1H), 4.38-4.25 (m, 1H), 3.45-3.38 (bs, 2H), 2.5-2.3 (m, 2H), 1.43-1.32 (m, 2H), 1.02-0.95 (m, 1H), 0.75-0.68 (m, 1H).
1H NMR (500MHz, DMSO-d6) " less important isomer ", δ 8.89 (s, 1H), 7.65-7.61 (m, 1H), 7.43 (d, 1H), 6.23-6.19 (m, 1H), 4.38-4.25 (m, 1H), 3.45-3.38 (bs, 2H), 2.5-2.3 (m, 2H), 1.43-1.32 (m, 2H), 1.02-0.95 (m, 1H), 0.75-0.68 (m, 1H).
Embodiment 21
N 1-(1-cyano group cyclopropyl)-N 2-(1S)-2,2-two fluoro-1-[4-(3-methyl-2-thienyl) phenyl] ethyl }-the L-leucine amide.
Figure A20048002452000821
Step 1:(2S)-and the 1-{[tertiary butyl (dimethyl) silyl] the oxygen base }-N-[(1Z)-2,2-difluoro ethylidene]-preparation of 4-pentane-2-amine
(2S)-1-{[tertiary butyl (dimethyl) silyl that will be in benzene] the oxygen base }-4-methylpentane-2-amine (embodiment 4 steps 1,8.5g, 36.8mmol) and difluoro acetaldehyde ethyl hemiacetal (5.0g, 39.7mmol) mixture refluxes with the Dean-stark trap and spends the night.Solvent is removed under vacuum.With resistates by short silica column with use hexane: EtOAc (10: 1) carries out wash-out, produces the title compound of light yellow oily.
1H?NMR(CD 3COCD 3)δ7.72(m，1H)，6.12(dt，1H)，3.70(dd，1H)，3.54(dd，1H)，3.36(m，1H)，1.48(m，2H)，1.32(m，1H)，0.95-0.78(m，15H)，0.06(s，3H)，0.02(s，3H).
Step 2:(2S)-1-{[(1S)-and 1-(4-bromophenyl)-2,2-two fluoro ethyls] amino }-preparation of 4-methylpentane-1-alcohol
With n-BuLi (the 2.5M hexane solution, 1.43mL) join-70 ℃ 1, in the THF solution of 4-dibromobenzene (884 milligrams), and mixture stirred 15 minutes.Then with (2S)-1-([tertiary butyl (dimethyl) silyl] oxygen base }-4-methyl-N-[(1E)-2,2-difluoro ethylidene] THF (8.5mL) solution of penta-2-amine (1.0g), and mixture stirred 1.5 hours.Under vigorous stirring, it is slowly poured in the ice-cold saturated ammonium chloride solution then.It is extracted with 3 parts of ethyl acetate.Organic layer salt water washing with merging obtains resistates with dried over mgso and vaporising under vacuum solvent, with this resistates at SiO 2On carry out purifying, use hexane and ethyl acetate gradient (90: 10～75: 25) elutriant, obtain title compound.To be dissolved in CH by the compound (200 milligrams) of last gained 3Among the CN (4mL), solution is cooled to 0 ℃.HF-pyridine (40 M) is dripped adding and made mixture reaction 16 hours.Be poured in the saturated sodium bicarbonate solution, acutely shake with the ethyl acetate adding with it.Organic layer is separated, and (2 * 50mL) aqueous layer extracted of further using ethyl acetate.With the organic layer salt water washing that merges, with dried over mgso with under vacuum solvent removed and to obtain resistates, with this resistates at SiO 2On carry out purifying, use hexane and ethyl acetate gradient (80: 20～60: 40) elutriant, obtain title compound.
1H?NMR(CD 3COCD 3)δ7.6(2H，d)，7.45(2H，d)，6.0(1H，dt)，4.25(1H，m)，3.65(1H，t)，3.5-3.55(1H，m)，3.3-3.35(1H，m)，2.55-2.65(1H，m)，2.15-2.25(1H，m)，1.6-1.7(1H，m)，1.3-1.4(1H，m)，1.2-1.3(1H，m)，0.9(3H，d)，0.8(3H，d).
Step 3:N-[(1S)-and 1-(4-bromophenyl)-2,2-two fluoro ethyls]-the leucic preparation of L-
With H 5IO 6/ CrO 3Suspension (the 0.40M CH of 5.5mL 3CN solution; Consult following note) be cooled to 0 ℃ and will derive from the CH of the alcohol (250mg) of step 2 3CN (3.7mL) solution drips and adds.Mixture was stirred 3.5 hours down at 0～5 ℃.Through after this stage, the 2.0mL oxygenant is added.1.5 after hour, under vigorous stirring, be poured into Na 2In the HPO4 buffered soln (containing 0.4g among the 10mL), (3 * 20mL) extract with ether with mixture.The ethereal extract water and the salt solution (1: 1) that merge are washed, use rare NaHSO 3Solution and salt water washing.Organic extract liquid is obtained resistates with dried over mgso, filtration with the solvent evaporate to dryness, can use without this resistates of purifying further.
Note: oxygenant (H 5IO 6/ CrO 3) as described in Tetrahedron Letters 39 (1998) 5323-5326, be prepared, but be to use the CH of HPLC level 3CN (containing 0.5% water); Do not add entry.
1H NMR (CD 3COCD 3) δ 7.55 (2H, d), 7.4 (2H, d), 6.05 (1H, dt), 3.95-4.05 (1H, m), 3.45 (1H, t), 2.7-3.0 (wide m, NH/OH), 1.85-1.95 (1H, m), 1.5 (2H, t), 0.95 (3H, d), 0.9 (3H, d).
Step 4:N 2-[(1S)-and 1-(4-bromophenyl)-2,2-two fluoro ethyls]-N 1The preparation of-(1-cyano group cyclopropyl)-L-leucine amide
In the DMF of the acid that derives from step 3 (258mg) (2mL) solution, add O-(7-azepine benzo triazol-1-yl)-N, N, N ', N '-tetramethyl-urea  hexafluorophosphate (337mg) and 1-amino-cyclopropane nitrile hydrochloride (175mg).Stir after 1 minute, stirred 16 hours with diisopropylethylamine (0.45mL) dropping adding with mixture.Be poured into that (3 * 15mL) extract in the saturated sodium bicarbonate aqueous solution and with ethyl acetate.With the extract salt water washing that merges, solvent is removed with dried over mgso with under vacuum.By chromatography at SiO 2Last use hexane and ethyl acetate (80: 20～50: 50) are carried out purifying to resistates.
1H?NMR(CD 3COCD 3)δ8.05(1H，m)，7.55(2H，d)，7.4(2H，d)，6.05(1H，dt)，3.95-4.05(1H，m)，3.25-3.3(1H，m)，2.4-2.45(1H，m)，1.8-1.9(1H，m)，1.4-1.55(2H，m)，0.95-1.1(2H，m)，0.95(6H，t).
Step 5:N 1-(1-cyano group cyclopropyl)-N 2-(1S)-2,2-two fluoro-1-[4-(4,4,5,5-tetramethyl--1,3,2-two oxa-boron heterocycle pentane-2-yls] phenyl] ethyl-preparation of L-leucine amide
In DMF (60mL) solution of the aromatic bromide that derives from step 4 (5.23g) and two (tetramethylethylene glycolization) two boron (3.8g), add potassium acetate (3.7g) and PdCl 2Dppf (309mg).Make nitrogen gas stream pass through suspension 1 minute.Reaction mixture is heated 16h down at 80 ℃.Make it be cooled to room temperature and change in the separating funnel.With saturated NaHCO 3Solution of (～120mL) and EtOAc (100mL) add.Organic layer is separated, and (2 * 50mL) extract water layer further to use 2 parts of EtOAc.With the organic layer that merges with the salt water washing, use Na 2SO 4Dry and concentrated.Thick material is carried out purifying (hexane/EtOAc is 80: 20～50: 50), the borate that obtains expecting on silica gel.
1H?NMR(CD 3COCD 3)δ8.15(bs，NH)，7.72(2H，d)，7.40(2H，d)，6.02(1H，dt)，3.95(1H，m)，3.25(1H，q)，2.38(1H，m)，1.72(1H，m)，1.27-1.50(16H，m)，0.85-1.05(8H，m).
Step 6:N 1-(1-cyano group cyclopropyl)-N 2The preparation of-{ (1S)-2,2-two fluoro-1-[4-(3-methyl-2-thienyl) phenyl] ethyl }-L-leucine amide.
In sealable microwave tube, make nitrogen gas stream pass through suspension 1 minute, this suspension is by the aromatic yl acid salt that derives from step 5 (200mg), 2-bromo-3 methyl thiophene (115mg), 2MNa 2CO 3(0.65mL), DMF (4.3mL) and PdCl 2Dppf (11mg) constitutes.Then mixture is heated 500 seconds (fixedly holding time: close) in microwave (SmithCreator), be heated to 120 ℃ of (absorption levels: height).It is cooled to room temperature, with ethyl acetate dilution be poured in the saturated sodium bicarbonate solution.Ethyl acetate layer is separated, and (2 * 15mL) aqueous layer extracted of further using ethyl acetate.With the acetic acid ethyl acetate extract that merges with the salt water washing with use dried over mgso.Removing desolvates obtains resistates, with this resistates by chromatography at SiO 2On carry out purifying, use hexane and ethyl acetate gradient (hexane/EtOAc is 80: 20～50: 50).
1H?NMR(CD 3COCD 3)δ8.13(bs，NH)，7.50(4H，s)，7.37(1H，d)，6.97(1H，d)，6.05(1H，t)，4.00(1H，m)，3.30(1H，m)，2.42(1H，m)，2.32(3H，s)，1.85(1H，m)，1.40-1.53(2H，m)，1.30-1.40(2H，m)，0.85-1.03(8H，m).
Embodiment 22
N 1-(1-cyano group tolyl)-N 2-{ (1S)-2,2,2-three fluoro-1-[4 '-((methylsulfonyl))-1,1 '-xenyl-4-yl] ethyl }-L-leucine amide synthetic
Figure A20048002452000851
To N-{ (1S)-2,2,2-three fluoro-1-[4 '-((methylsulfonyl))-1; 1 '-xenyl-4-yl] ethyl }-L-leucine (embodiment 4 steps 6; 21mg, and adding 2-phenylamino acetonitrile hydrochloride in DMF mixture 0.046mmol) (8.6mg, 0.051mmol).With N-methylmorpholine (41 μ L, 0.368mmol) and the N-propyl phosphonous acid acid anhydride of the tripolymer point 50% of cyclisation (55 μ L, 0.092mmol) order adds, and the mixture stirring is spent the night.Volatile matter obtains evaporation in GenevacHT-4.Resistates is dissolved in CH 2Cl 2(5mL), filter by SiOH SPE tube (500mg) with BTMA carbonate silica gel treatment 30 minutes with it.With macroporous resin A-21 treatment soln, and refilter once.With solution concentration, obtain 1: 1 epimer mixture.
MS(-ESI)：556.0[M-1] -
Embodiment 23
N 1-(cyano group cyclopropyl)-N 2-{ (1S)-2,2-two fluoro-1-[2 ', 4 '-two fluoro-1,1 '-xenyl-4-yl] ethyl }-L-leucine amide synthetic
Figure A20048002452000861
Use embodiment 21 described steps, the 2-bromo-3 methyl thiophene in the step 6 is wherein replaced with 1-bromo-2, the 4-phenyl-difluoride obtains the title compound of white solid.
MS(+API)：448.1[M+1] +.
Embodiment 24
Step 4:3-oxo-4-[(N-{ (1S)-2,2,2-three fluoro-1-[4 '-((methylsulfonyl)) xenyl-4-yl] ethyl }-the L-leucyl) amino] nitrogen heterocyclic heptane-1-carboxylic acid benzyl ester synthetic
Figure A20048002452000862
Step 1:3-hydroxyl-4-[(N-{ (1S)-2,2,2-three fluoro-1-[4 '-((methylsulfonyl)) xenyl-4-yl] ethyl }-the L-leucyl) amino] azepan-1-carboxylic acid benzyl ester
To N-{ (1S)-2; 2; 2-three fluoro-1-[4 '-((methylsulfonyl))-1; 1 '-xenyl-4-yl] ethyl }-L-leucine (embodiment 4 steps 6; 605mg, 1.37mmol), 4-amino-3-hydroxyl azepan-(WO 0134565, J.Med.Chem. for 1-carboxylic acid benzyl ester; 44; 1380,2001,326mg; 1.23mmol) and PyBOP (724mg; 1.39mmol) 10mL DMF solution in add triethylamine (0.45mL 3.2mmol), stir 1h with mixture; be warming up to room temperature 1h, then at NaHCO 3And distribute between the ether.Is the washing of 3.5 phosphate buffered saline buffer with organic phase with the pH value, uses the salt water washing then, uses MgSO 4Dry.(gradient is 65% ethyl acetate: hexane～100% ethyl acetate) carry out purifying, the title compound as isomer mixt is provided by silica gel chromatography.
Step 2:3-oxo-4-[(N-{ (1S)-2,2,2-three fluoro-1-[4 '-((methylsulfonyl)) xenyl-4-yl] ethyl }-the L-leucyl) amino] azepan-1-carboxylic acid benzyl ester
3-hydroxyl-4-[(N-{ (1S)-2 to 0 ℃; 2; 2-three fluoro-1-[4 '-((methylsulfonyl)) xenyl-4-yl] ethyl }-the L-leucyl) amino] (98mg adds Dess-Martin and crosses iodine alkane (periodinane) azepan-1-carboxylic acid benzyl ester in methylene dichloride 0.14mmol) (3mL) solution.Mixture is warming up to room temperature, stirs 15h, between ethyl acetate and 1M NaOH, distribute then.With organic phase with the salt water washing with use MgSO 4Dry.(gradient is 40%～70% ethyl acetate: hexane) carry out purifying, the title compound as non-corresponding isomer mixture is provided by silica gel chromatography.
MS(+ESI)：688.4[M+1] +
Embodiment 25
N 1-[3-oxo-1-(pyridine-2-base alkylsulfonyl) azepan-4-yl]-N 2-{ (1S)-2,2,2-three fluoro-1-[4 '-((methylsulfonyl)) xenyl-4-yl] ethyl }-L-leucine amide synthetic
Figure A20048002452000871
Step 1:N 1-[3-hydroxyl azepan-4-yl]-N 2-(1S)-2,2,2-three fluoro-1-[4 '-((methylsulfonyl)) xenyl-4-yl] ethyl }-the L-leucine amide
Make benzyl 3-hydroxyl-4-[(N-{ (1S)-2; 2; 2-three fluoro-1-[4 '-((methylsulfonyl)) xenyl-4-yl] ethyl }-the L-leucyl) amino] azepan-1-carboxylicesters (embodiment 24 steps 1; 710mg; 1.03mmol) and 2: 1 EtOH of 10% Pd/C (490mg): EtOAc (80mL) solution is full of hydrogen and stirs 2h under the hydrogen bubbling.With reaction mixture filtration over celite and concentrating, produce title compound.
Step 2:N 1-[3-hydroxyl-1-(pyridine-2-base alkylsulfonyl) azepan-4-yl]-N 2-(1S)-2,2,2-three fluoro-1-[4 '-((methylsulfonyl)) xenyl-4-yl] ethyl }-the L-leucine amide
To 0 ℃ N 1-(3-hydroxyl azepan-4-yl)-N 2-{ (1S)-2; 2,2-three fluoro-1-[4 '-((methylsulfonyl)) xenyl-4-yl] ethyl }-(567mg adds triethylamine (0.25mL in methylene dichloride 1.02mmol) (10mL) solution to the L-leucine amide; 1.8mmol) and 2-pyridine SULPHURYL CHLORIDE (204mg, 1.15mmol).Mixture is warming up to room temperature keeps 1h, then with it at methylene dichloride and NaHCO 3Between distribute.With organic phase salt water washing, the filtration cotton yarn also concentrates.(gradient is 70% ethyl acetate: hexane～100% ethyl acetate) carry out purifying, obtain the title compound as isomer mixt by silica gel chromatography.
Step 3:N 1-[3-oxo-1-(pyridine-2-base alkylsulfonyl) azepan-4-yl]-N 2-(1S)-2,2,2-three fluoro-1-[4 '-((methylsulfonyl)) xenyl-4-yl] ethyl }-the L-leucine amide
To room temperature N 1-[3-hydroxyl-1-(pyridine-2-base alkylsulfonyl) azepan-4-yl]-N 2-(1S)-2,2,2-three fluoro-1-[4 '-((methylsulfonyl)) xenyl-4-yl] ethyl }-(460mg adds Dess-Martin and crosses iodine alkane the L-leucine amide in methylene dichloride 0.73mmol) (15mL) solution.Mixture is stirred 1h, with the methylene dichloride dilution,, use the salt water washing then then with the 1MNaOH washing.Organic phase is filtered cotton yarn and concentrates.(gradient is 60% ethyl acetate: hexane～100% ethyl acetate) carry out purifying, obtain the title compound as non-corresponding isomer mixture by silica gel chromatography.
MS(+ESI)：695.3[M+1] +
Embodiment 26
N 2-{ (4-bromophenyl) (4-p-methoxy-phenyl) methyl }-N 1Synthesizing of-(cyanogen methyl)-L-leucine amide
Step 1:(4-bromophenyl) (4-methoxyphenyl) methyl alcohol
With 1, (9.1g, THF 38mmol) (80mL) solution is cooled to-78 ℃ to the 4-dibromobenzene, n-Butyl Lithium (16mL, 2.5M hexane solution) is dripped add.After 15 minutes, aubepine (5g, 37mmol is in 4.5mL THF) is dripped adding.Again through after 20 minutes, with reaction mixture with methyl alcohol (5mL) and saturated aqueous ammonium chloride (100mL) quencher.Reaction mixture is extracted with three parts of 100mL ethyl acetate, and with the organic layer salt water washing (50mL) that merges.With dried over mgso organic layer and under reduced pressure concentrated, produce title compound, can be directly used in next step.
Step 2:N-[(4-bromophenyl) (4-methoxyphenyl) methyl]-L-leucine methyl esters
With (4-bromophenyl) (4-methoxyphenyl) methyl alcohol (1.15g, 3.9mmol) with four fourth brometo de amonios (130mg 0.4mmol) is dissolved in the methylene dichloride (4mL) together.Then 48% hydrobromic acid aqueous solution (3.3mL) is added, with reaction mixture vigorous stirring 40 hours.Product is distributed between water (20mL) and methylene dichloride (30mL) and carry out drying with sal epsom.Organic layer is concentrated into about 10mL and L-leucine methyl esters (free alkali) is added with the form of methylene dichloride (20mL) solution, succeeded by triethylamine (3mL) is added.Reaction mixture is stirred 20 minutes (white precipitate occurring) down at 35 ℃.In ether (50mL) and water (30mL), reaction is adjusted.Be separated usefulness saturated aqueous ammonium chloride (30mL) and salt solution (30mL) washing organic layer with two.With dried over mgso with after under reduced pressure concentrating, prepare N-(4-bromophenyl) (4-methoxyphenyl) methyl]-L-leucine methyl esters, and on silica gel, use 10% ethyl acetate, 90% hexane to carry out purifying.
Step 3:N-[(4-bromophenyl) (4-methoxyphenyl) methyl]-the L-leucine
The N-[(4-bromophenyl in about 2: 1: 1 THF/MeOH/ water to the 60mL under the room temperature) (4-methoxyphenyl) methyl]-L-leucine methyl esters (1.25g, 3mmol) in the solution, add lithium hydroxide monohydrate (250mg, 6mmol).The stirred overnight mixture also concentrates.Be to distribute between 3.5 the phosphate buffered saline buffer (50mL) resistates in methylene dichloride (50mL) and pH value.With aqueous phase separation with two parts of 50mL washed with dichloromethane.Organic phase with dried over sodium sulfate with under reduced pressure concentrate, is obtained a kind of solid, and the grinding in the cold methylene dichloride of minute quantity of this solid obtains the N-[(4-bromophenyl) (4-methoxyphenyl) methyl]-the L-leucine.
Step 4:N 2-[(4-bromophenyl) (4-methoxyphenyl) methyl]-N 1-(cyanogen methyl)-L-leucine amide
With the N-[(4-bromophenyl) (4-methoxyphenyl) methyl]-(149mg, 0.37mmol) (87mg, mixture 0.73mmol) is dissolved in the 5mL dimethyl formamide L-leucine with the aminoacetonitriles hydrochloride.With HATU (153mg, 0.403mmol) disposable adding, add then triethylamine (0.18mL, 1.31mmol).Mixture stirred spend the night, be poured into pH value then and be 4 phosphate buffered saline buffer (40mL) and neutralize and extract with ethyl acetate (50mL).With three parts of 50mL water washing organic phases, with dried over sodium sulfate and under reduced pressure concentrated.Purifying on silica gel (30% ethyl acetate/hexane) provides N 2-[(4-bromophenyl) (4-methoxyphenyl) methyl]- N 1-(cyanogen methyl)-L-leucine amide.
MS(+ESI)：443.9[M+1].
The kethepsin combination
Each embodiment obeys following process among the embodiment 1～26: in the model that computer generates, compound at the cathepsin K reactive site by energy minimization.The model that the cathepsin K computer generates is based on the crystalline structure of Protein Data Bank inlet 1MEM, hydrogen has been joined on this crystalline structure, but when energy minimization begins, do not carried out non-hydrogen energy minimization (so protein side chain keeps the geometric configuration of x-ray crystal structure).The bonding position of compound is determined according to following imagination: 1) covalent linkage is at S 1And be marked as in the compound between the close electrical carbon atom of " 1 " and formed.In conjunction with being used for chemical formula of the present invention, this has determined corresponding to R 1And R 2Molecule fragment; 2) for the compound segment corresponding to chemical formula of the present invention, hydrogen atom on the amine and the Sauerstoffatom on the Gly66 form hydrogen bond, and therefore the distance between these two atoms is less than 4A; 3) corresponding to R 2And R 2The compound fragment, according to the mark carbon atom on the compound chemical structure, be in kethepsin sublocus S respectively 2And S 3In.For example, the carbon atom that is marked as " 2 " in the chemical structure is positioned at S 2In, and the carbon atom that is labeled as " 3 " is positioned at S 3In, make the distance of the α of cathepsin C in the table of distances 1 like this in table 1 provides a dust scope of distance.Though these placements are artificial and approximate operations, and they are placed wittingly to produce favourable interaction.For synthetic compound, be used to calculate corresponding to the enantiomer of chemical formula of the present invention as racemic mixture.Use the software MacroModel and the MMFFs field of force, energy minimization is carried out.All atoms of compound all are allowed to move, but for cathepsin K, the protein side chain that only has the atom in compound 6  scopes allows to move; In this case, whole side chain can move.The default parameter that is used for energy minimization is selected, and simultaneously continuous solvent is corresponding to water.The result of energy minimization is favourable for compound: do not have bad steric interaction between part and cathepsin K reactive site, and the favourable interaction between reactive site and the part (oleophylic interacts and hydrogen bonded) is confirmed by the distance that provides in the table 1.The distance that table 1 provides is taken from as mentioned above the part-enzyme title complex of the energy minimization that is formed by compound and cathepsin K.The hurdle that is labeled as " Gly66 hydrogen bond " has provided the hydrogen that is formed on the compound amine and the distance of the hydrogen bond between the oxygen on the Gly 66.R in the table 1 2Under three hurdles all be labeled as title with C α corresponding to a C α in the kethepsin; These hurdles have provided the distance between the carbon atom that is labeled as " 2 " in kethepsin in the C α that indicates and the compound structure.Similarly, " R in the table 1 2" under two hurdles provided distance between the carbon atom that is labeled as " 3 " in the C α that in kethepsin, indicates and the compound structure.Be formed at the covalent linkage between the close electrical carbon atom that is labeled as " 1 " in the sulphur of the halfcystine of residue 25 in the kethepsin and this compound structure, guaranteed with this compound structure in be labeled as distance＜5  of the carbon atom of " 1 ".Thus, embodiment 1～26 satisfies described in this article criterion distance.
The distance (representing) of the atom of the atom of table 1. from cathepsin K to the embodiment 1～26 with .Consult text.
Embodiment R2 R3
The Gly66 hydrogen bond Cα 26 Cα 68 Cα 134 Cα 66 Cα 60
1 2.4 6.4 7.7 5.5 4.5 6.1
2 2.5 6.4 7.7 5.5 4.4 5.9
3 2.5 6.5 7.7 5.5 4.8 6.3
4 2.6 6.4 7.7 5.6 4.8 6.3
5 2.6 6.4 7.7 5.4 4.8 6.3
6 2.5 6.4 7.7 5.5 4.7 6.3
7 2.5 6.4 7.7 5.5 4.6 6.1
8 2.5 6.5 7.6 5.4 4.7 6.3
9 2.4 6.5 7.8 5.3 4.6 6.2
10 2.5 6.8 8.0 5.4 4.6 6.1
11 2.6 6.6 7.7 5.4 4.8 6.3
12 2.6 6.2 7.7 5.6 4.8 6.5
13 2.7 6.7 7.9 5.6 5.0 6.5
14 2.5 6.5 7.7 5.5 4.5 6.0
15 2.5 6.5 7.7 5.4 4.7 6.3
16 2.4 6.5 7.5 5.4 4.7 6.2
17 2.4 6.6 7.5 5.4 4.7 6.2
18 2.4 6.4 7.5 5.4 4.7 6.2
19 2.5 6.7 7.5 5.4 4.8 6.2
20 2.3 6.4 7.5 5.5 4.5 6.1
21 2.4 6.4 7.6 5.4 4.6 6.2
22 2.5 6.5 7.6 5.5 4.7 6.2
23 2.4 6.4 7.6 5.4 4.7 6.3
24 2.0 6.7 7.3 5.4 4.6 6.0
25 2.4 6.5 7.6 5.4 4.6 6.2
26 2.4 6.4 7.7 5.5 4.8 6.4
The enzyme test of purifying
Be disclosed in compound among the application and demonstrate activity in testing below.In addition, the compound that is disclosed in the application has the enhanced pharmacological profile with respect to previous disclosed compound.
The cathepsin K test
Prepare test compound in dimethyl sulfoxide (DMSO) (DMSO) from the serial dilution thing of 500 μ M up to 0.0085 μ M.The DMSO that then 2 μ L is taken from each dilution joins 50 μ L test damping fluid (MES, 50mM (pH value 5.5); EDTA, 2.5mM; DTT, 2.5mM and 10%DMSO)) and the human tissue Proteinase K (0.4nM) of 25 μ L in test buffered soln in.Testing liquid was mixed for 5～10 seconds and at room temperature cultivated 15 minutes on swing plate.To join in the evaluation and test solution at the Z-Leu-Arg-AMC (8 μ M) in the 25 μ L evaluation and test damping fluid.By spectrofluorimetry (Ex λ=355nm; Em λ=460nm), the hydrolysis of tonka bean camphor leavings group (AMC) was followed the tracks of 10 minutes.By with the experimental value match to about the standard mathematical model of dose response curve, calculate restraining effect per-cent.
Cathepsin L's test
Prepare test compound in dimethyl sulfoxide (DMSO) (DMSO) from the serial dilution thing (1/3) of 500 μ M until 0.0085 μ M.The DMSO that then 2 μ L is taken from each dilution joins 50 μ L test damping fluid (MES, 50mM (pH value 5.5); EDTA, 2.5mM; DTT, 2.5mM and 10%DMSO) and the human tissue proteolytic enzyme L (0.5nM) of 25 μ L in the test buffer soln in.Testing liquid was mixed for 5～10 seconds on swing plate also at room temperature cultivated 15 minutes.To join in the evaluation and test solution at the Z-Leu-Arg-AMC (8 μ M) in the 25 μ L evaluation and test damping fluid.By spectrofluorimetry (Ex λ=355nm; Em λ=460nm), the hydrolysis of tonka bean camphor leavings group (AMC) was followed the tracks of 10 minutes.By with the experimental value match to about the standard mathematical model of dose response curve, calculate restraining effect per-cent.
Cathepsin B's test
Prepare test compound in dimethyl sulfoxide (DMSO) (DMSO) in from the serial dilution thing (1/3) of 500 μ M to 0.0085 μ M.The DMSO that then 2 μ L is taken from each dilution joins 50 μ L test damping fluid (MES, 50mM (pH value 5.5); EDTA, 2.5mM; DTT, 2.5mM and 10%DMSO) and the human tissue proteolytic enzyme B (4.0nM) of 25 μ L in the test buffer soln in.Testing liquid was mixed for 5～10 seconds on swing plate also at room temperature cultivated 15 minutes.To join in the evaluation and test solution at the Z-Leu-Arg-AMC (8 μ M) in the 25 μ L evaluation and test damping fluid.By spectrofluorimetry (Ex λ=355nm; Em λ=460nm), the hydrolysis of tonka bean camphor leavings group (AMC) was followed the tracks of 10 minutes.By with the experimental value match to about the standard mathematical model of dose response curve, calculate restraining effect per-cent.
The cathepsin S test
Prepare test compound serial dilution thing (1/3) from 500 μ M to 0.0085 μ M in dimethyl sulfoxide (DMSO) (DMSO).The DMSO that then 2 μ L is taken from each dilution joins 50 μ L test damping fluid (MES, 50mM (pH value 5.5); EDTA, 2.5mM; DTT, 2.5mM and 10%DMSO) and the human tissue proteolytic enzyme S (20nM) of 25 μ L in the test buffer soln in.Testing liquid was mixed for 5～10 seconds and at room temperature cultivated 15 minutes on swing plate.To join in the evaluation and test solution at the Z-Leu-Arg-AMC (8 μ M) in the 25 μ L evaluation and test damping fluid.By spectrofluorimetry (Ex λ=355nm; Em λ=460nm), the hydrolysis of tonka bean camphor leavings group (AMC) was followed the tracks of 10 minutes.By with the experimental value match to about the standard mathematical model of dose response curve, calculate restraining effect per-cent.
Sequence table
<110>Merck?Frosst.
Bayly，Christopher
Black.Cameron
McKay，Daniel
＜120〉cathepsin inhibitors
<130>MCO78Y
<150>60/498,017
<151>2003-08-27
<160>13
<170>FastSEQ?for?Windows?Version?4.0
<210>1
<211>215
<212>PRT
＜213〉people
<400>1
Ala?Pro?Asp?Ser?Val?Asp?Tyr?Arg?Lys?Lys?Gly?Tyr?Val?Thr?Pro?Val
1 5 10 15
Lys?Asn?Gln?Gly?Gln?Cys?Gly?Ser?Cys?Trp?Ala?Phe?Ser?Ser?Val?Gly
20 25 30
Ala?Leu?Glu?Gly?Gln?Leu?Lys?Lys?Lys?Thr?Gly?Lys?Leu?Leu?Asn?Leu
35 40 45
Ser?Pro?Gln?Asn?Leu?Val?Asp?Cys?Val?Ser?Glu?Asn?Asp?Gly?Cys?Gly
50 55 60
Gly?Gly?Tyr?Met?Thr?Asn?Ala?Phe?Gln?Tyr?Val?Gln?Lys?Asn?Arg?Gly
65 70 75 80
Ile?Asp?Ser?Glu?Asp?Ala?Tyr?Pro?Tyr?Val?Gly?Gln?Glu?Glu?Ser?Cys
85 90 95
Met?Tyr?Asn?Pro?Thr?Gly?Lys?Ala?Ala?Lys?Cys?Arg?Gly?Tyr?Arg?Glu
100 105 110
Ile?Pro?Glu?Gly?Asn?Glu?Lys?Ala?Leu?Lys?Arg?Ala?Val?Ala?Arg?Val
115 120 125
Gly?Pro?Val?Ser?Val?Ala?Ile?Asp?Ala?Ser?Leu?Thr?Ser?Phe?Gln?Phe
130 135 140
Tyr?Ser?Lys?Gly?Val?Tyr?Tyr?Asp?Glu?Ser?Cys?Asn?Ser?Asp?Asn?Leu
145 150 155 160
Asn?His?Ala?Val?Leu?Ala?Val?Gly?Tyr?Gly?Ile?Gln?Lys?Gly?Asn?Lys
165 170 175
His?Trp?Ile?Ile?Lys?Asn?Ser?Trp?Gly?Glu?Asn?Trp?Gly?Asn?Lys?Gly
180 185 190
Tyr?Ile?Leu?Met?Ala?Arg?Asn?Lys?Asn?Asn?Ala?Cys?Gly?Ile?Ala?Asn
195 200 205
Leu?Ala?Ser?Phe?Pro?Lys?Met
210 215
<210>2
<211>254
<212>PRT
＜213〉people
<400>2
Leu?Pro?Ala?Ser?Phe?Asp?Ala?Arg?Glu?Gln?Trp?Pro?Gln?Cys?Pro?Thr
1 5 10 15
Ile?Lys?Glu?Ile?Arg?Asp?Gln?Gly?Ser?Cys?Gly?Ser?Cys?Met?Ala?Phe
20 25 30
Gly?Ala?Val?Glu?Ala?Ile?Ser?Asp?Arg?Ile?Cys?Ile?His?Thr?Asn?Ala
35 40 45
His?Val?Ser?Val?Glu?Val?Ser?Ala?Glu?Asp?Leu?Leu?Thr?Cys?Cys?Gly
50 55 60
Ser?Met?Cys?Gly?Asp?Gly?Cys?Asn?Gly?Gly?Tyr?Pro?Ala?Glu?Ala?Trp
65 70 75 80
Asn?Phe?Trp?Thr?Arg?Lys?Gly?Leu?Val?Ser?Gly?Gly?Leu?Tyr?Glu?Ser
85 90 95
His?Val?Gly?Cys?Arg?Pro?Tyr?Ser?Ile?Pro?Pro?Cys?Glu?His?His?Val
100 105 110
Asn?Gly?Ser?Arg?Pro?Pro?Cys?Thr?Gly?Glu?Gly?Asp?Thr?Pro?Lys?Cys
115 120 125
Ser?Lys?Ile?Cys?Glu?Pro?Gly?Tyr?Ser?Pro?Thr?Tyr?Lys?Gln?Asp?Lys
130 135 140
His?Tyr?Gly?Tyr?Asn?Ser?Tyr?Ser?Val?Ser?Asn?Ser?Glu?Lys?Asp?Ile
145 150 155 160
Met?Ala?Glu?Ile?Tyr?Lys?Asn?Gly?Pro?Val?Glu?Gly?Ala?Phe?Ser?Val
165 170 175
Tyr?Ser?Asp?Phe?Leu?Leu?Tyr?Lys?Ser?Gly?Val?Tyr?Gln?His?Val?Thr
180 185 190
Gly?Glu?Met?Met?Gly?Gly?His?Ala?Ile?Arg?Ile?Leu?Gly?Trp?Gly?Val
195 200 205
Glu?Asn?Gly?Thr?Pro?Tyr?Trp?Leu?Val?Ala?Asn?Ser?Trp?Asn?Thr?Asp
210 215 220
Trp?Gly?Asp?Asn?Gly?Phe?Phe?Lys?Ile?Leu?Arg?Gly?Gln?Asp?His?Cys
225 230 235 240
GlyIle?Glu?Ser?Glu?Val?Val?Ala?Gly?Ile?Pro?Arg?Thr?Asp
245 250
<210>3
<211>214
<212>PRT
＜213〉people
<400>3
Ala?Pro?Pro?Glu?Trp?Asp?Trp?Arg?Ser?Lys?Gly?Ala?Val?Thr?Lys?Val
1 5 10 15
Lys?Asp?Gln?Gly?Met?Cys?Gly?Ser?Cys?Trp?Ala?Phe?Ser?Val?Thr?Gly
20 25 30
Asn?Val?Glu?Gly?Gln?Trp?Phe?Leu?Asn?Gln?Gly?Thr?Leu?Leu?Ser?Leu
35 40 45
Ser?Glu?Gln?Glu?Leu?Leu?Asp?Cys?Asp?Lys?Met?Asp?Lys?Ala?Cys?Met
50 55 60
Gly?Gly?Leu?Pro?Ser?Asn?Ala?Tyr?Ser?Ala?Ile?Lys?Asn?Leu?Gly?Gly
65 70 75 80
Leu?Glu?Thr?Glu?Asp?Asp?Tyr?Ser?Tyr?Gln?Gly?His?Met?Gln?Ser?Cys
85 90 95
Asn?Phe?Ser?Ala?Glu?Lys?Ala?Lys?Val?Tyr?Ile?Asn?Asp?Ser?Val?Glu
100 105 110
Leu?Ser?Gln?Asn?Glu?Gln?Lys?Leu?Ala?Ala?Trp?Leu?Ala?Lys?Arg?Gly
115 120 125
Pro?Ile?Ser?Val?Ala?Ile?Asn?Ala?Phe?Gly?Met?Gln?Phe?Tyr?Arg?His
130 135 140
Gly?Ile?Ser?Arg?Pro?Leu?Arg?Pro?Leu?Cys?Ser?Pro?Trp?Leu?Ile?Asp
145 150 155 160
His?Ala?Val?Leu?Leu?Val?Gly?Tyr?Gly?Asn?Arg?Ser?Asp?Val?Pro?Phe
165 170 175
Trp?Ala?Ile?Lys?Asn?Ser?Trp?Gly?Thr?Asp?Trp?Gly?Glu?Lys?Gly?Tyr
180 185 190
Tyr?Tyr?Leu?His?Arg?Gly?Ser?Gly?Ala?Cys?Gly?Val?Asn?Thr?Met?Ala
195 200 205
Ser?Ser?Ala?Val?Val?Asp
210
<210>4
<211>220
<212>PRT
＜213〉people
<400>4
Tyr?Pro?Pro?Ser?Val?Asp?Trp?Arg?Lys?Lys?Gly?Asn?Phe?Val?Ser?Pro
1 5 10 15
Val?Lys?Asn?Gln?Gly?Ala?Cys?Gly?Ser?Cys?Trp?Thr?Phe?Ser?Thr?Thr
20 25 30
Gly?Ala?Leu?Glu?Ser?Ala?Ile?Ala?Ile?Ala?Thr?Gly?Lys?Met?Leu?Ser
35 40 45
Leu?Ala?Glu?Gln?Gln?Leu?Val?Asp?Cys?Ala?Gln?Asp?Phe?Asn?Asn?His
50 55 60
Gly?Cys?Gln?Gly?Gly?Leu?Pro?Ser?Gln?Ala?Phe?Glu?Tyr?Ile?Leu?Tyr
65 70 75 80
Asn?Lys?Gly?Ile?Met?Gly?Glu?Asp?Thr?Tyr?Pro?Tyr?Gln?Gly?Lys?Asp
85 90 95
Gly?Tyr?Cys?Lys?Phe?Gln?Pro?Gly?Lys?Ala?Ile?Gly?Phe?Val?Lys?Asp
100 105 110
Val?Ala?Asn?Ile?Thr?lle?Tyr?Asp?Glu?Glu?Ala?Met?Val?Glu?Ala?Val
115 120 125
Ala?Leu?Tyr?Asn?Pro?Val?Ser?Phe?Ala?Phe?Glu?Val?Thr?Gln?Asp?Phe
130 135 140
Met?Met?Tyr?Arg?Thr?Gly?Ile?Tyr?Ser?Ser?Thr?Ser?Cys?His?Lys?Thr
145 150 155 160
Pro?Asp?Lys?Val?Asn?His?Ala?Val?Leu?Ala?Val?Gly?Tyr?Gly?Glu?Lys
165 170 175
Asn?Gly?Ile?Pro?Tyr?Trp?Ile?Val?Lys?Asn?Ser?Trp?Gly?Pro?Gln?Trp
180 185 190
Gly?Met?Asn?Gly?Tyr?Phe?Leu?Ile?Glu?Arg?Gly?Lys?Asn?Met?Cys?Gly
195 200 205
Leu?Ala?Ala?Cys?Ala?Ser?Tyr?Pro?Ile?Pro?Leu?Val
210 215 220
<210>5
<211>220
<212>PRT
＜213〉people
<400>5
Ala?Pro?Arg?Ser?Val?Asp?Trp?Arg?Glu?Lys?Gly?Tyr?Val?Thr?Pro?Val
1 5 10 15
Lys?Asn?Gln?Gly?Gln?Cys?Gly?Ser?Cys?Trp?Ala?Phe?Ser?Ala?Thr?Gly
20 25 30
Ala?Leu?Glu?Gly?Gln?Met?Phe?Arg?Lys?Thr?Gly?Arg?Leu?Ile?Ser?Leu
35 40 45
Ser?Glu?Gln?Asn?Leu?Val?Asp?Cys?Ser?Gly?Pro?Gln?Gly?Asn?Glu?Gly
50 55 60
Cys?Asn?Gly?Gly?Leu?Met?Asp?Tyr?Ala?Phe?Gln?Tyr?Val?Gln?Asp?Asn
65 70 75 80
Gly?Gly?Leu?Asp?Ser?Glu?Glu?Ser?Tyr?Pro?Tyr?Glu?Ala?Thr?Glu?Glu
85 90 95
Ser?Cys?Lys?Tyr?Asn?Pro?Lys?Tyr?Ser?Val?Ala?Asn?Asp?Thr?Gly?Phe
100 105 110
Val?Asp?Ile?Pro?Lys?Gln?Glu?Lys?Ala?Leu?Met?Lys?Ala?Val?Ala?Thr
115 120 125
Val?Gly?Pro?Ile?Ser?Val?Ala?Ile?Asp?Ala?Gly?His?Glu?Ser?Phe?Leu
130 135 140
Phe?Tyr?Lys?Glu?Gly?Ile?Tyr?Phe?Glu?Pro?Asp?Cys?Ser?Ser?Glu?Asp
145 150 155 160
Met?Asp?His?Gly?Val?Leu?Val?Val?Gly?Tyr?Gly?Phe?Glu?Ser?Thr?Glu
165 170 175
Ser?Asp?Asn?Asn?Lys?Tyr?Trp?Leu?Val?Lys?Asn?Ser?Trp?Gly?Glu?Glu
180 185 190
Trp?Gly?Met?Gly?Gly?Tyr?Val?Lys?Met?Ala?Lys?Asp?Arg?Arg?Asn?His
195 200 205
Cys?Gly?Ile?Ala?Ser?Ala?Ala?Ser?Tyr?Pro?Thr?Val
210 215 220
<210>6
<211>214
<212>PRT
＜213〉people
<400>6
Leu?Pro?Leu?Arg?Phe?Asp?Trp?Arg?Asp?Lys?Gln?Val?Val?Thr?Gln?Val
1 5 10 15
Arg?Asn?Gln?Gln?Met?Cys?Gly?Gly?Cys?Trp?Ala?Phe?Ser?Val?Val?Gly
20 25 30
Ala?Val?Glu?Ser?Ala?Tyr?Ala?Ile?Lys?Gly?Lys?Pro?Leu?Glu?Asp?Leu
35 40 45
Ser?Val?Gln?Gln?Val?Ile?Asp?Cys?Ser?Tyr?Asn?Asn?Tyr?Gly?Cys?Asn
50 55 60
Gly?Gly?Ser?Thr?Leu?Asn?Ala?Leu?Asn?Trp?Leu?Asn?Lys?Met?Gln?Val
65 70 75 80
Lys?Leu?Val?Lys?Asp?Ser?Glu?Tyr?Pro?Phe?Lys?Ala?Gln?Asn?Gly?Leu
85 90 95
Cys?His?Tyr?Phe?Ser?Gly?Ser?His?Ser?Gly?Phe?Ser?Ile?Lys?Gly?Tyr
100 105 110
Ser?Ala?Tyr?Asp?Phe?Ser?Asp?Gln?Glu?Asp?Glu?Met?Ala?Lys?Ala?Leu
115 120 125
Leu?Thr?Phe?Gly?Pro?Leu?Val?Val?Ile?Val?Asp?Ala?Val?Ser?Trp?Gln
130 135 140
Asp?Tyr?Leu?Gly?Gly?Ile?Ile?Gln?His?His?Cys?Ser?Ser?Gly?Glu?Ala
145 150 155 160
Asn?His?Ala?Val?Leu?Ile?Thr?Gly?Phe?Asp?Lys?Thr?Gly?Ser?Thr?Pro
165 170 175
Tyr?Trp?Ile?Val?Arg?ASn?Ser?Trp?Gly?Ser?SeT?Trp?Gly?Val?Asp?Gly
180 185 190
Tyr?Ala?His?Val?Lys?Met?Gly?Ser?Asn?Val?Cys?Gly?Ile?Ala?Asp?Ser
195 200 205
Val?Ser?Ser?Ile?Phe?Val
210
<210>7
<211>217
<212>PRT
＜213〉people
<400>7
Leu?Pro?Asp?Ser?Val?Asp?Trp?Arg?Glu?Lys?Gly?Cys?Val?Thr?Glu?Val
1 5 10 15
Lys?Tyr?Gln?Gly?Ser?Cys?Gly?Ala?Cys?Trp?Ala?Phe?Ser?Ala?Val?Gly
20 25 30
Ala?Leu?Glu?Ala?Gln?Leu?Lys?Leu?Lys?Thr?Gly?Lys?Leu?Val?Thr?Leu
35 40 45
Ser?Ala?Gln?Asn?Leu?Val?Asp?Cys?Ser?Thr?Glu?Lys?Tyr?Gly?ASn?Lys
50 55 60
Gly?Cys?Asn?Gly?Gly?Phe?Met?Thr?Thr?Ala?Phe?Gln?Tyr?Ile?Ile?Asp
65 70 75 80
Asn?Lys?Gly?Ile?Asp?Ser?Asp?Ala?Ser?Tyr?Pro?Tyr?Lys?Ala?Met?Asp
85 90 95
Gln?Lys?Cys?Gln?Tyr?Asp?Ser?Lys?Tyr?Arg?Ala?Ala?Thr?Cys?Ser?Lys
100 105 110
Tyr?Thr?Glu?Leu?Pro?Tyr?Gly?Arg?Glu?Asp?Val?Leu?Lys?Glu?Ala?Val
115 120 125
Ala?ASn?Lys?Gly?Pro?Val?Ser?Val?Gly?Val?Asp?Ala?Arg?His?Pro?Ser
130 135 140
Phe?Phe?Leu?Tyr?Arg?Ser?Gly?Val?Tyr?Tyr?Glu?Pro?Ser?Cys?Thr?Gln
145 150 155 160
Asn?Val?Asn?His?Gly?Val?Leu?Val?Val?Gly?Tyr?Gly?Asp?Leu?Asn?Gly
165 170 175
Lys?Glu?Tyr?Trp?Leu?Val?Lys?Asn?Ser?Trp?Gly?His?Asn?Phe?Gly?Glu
180 185 190
Glu?Gly?Tyr?Ile?Arg?Met?Ala?Arg?Asn?Lys?Gly?Asn?His?Cys?Gly?Ile
195 200 205
Ala?Ser?Phe?Pro?Ser?Tyr?Pro?Glu?Ile
210 215
<210>8
<211>249
<212>PRT
＜213〉people
<400>8
Val?Pro?Phe?Ser?Cys?Asp?Trp?Arg?Lys?Val?Ala?Gly?Ala?Ile?Ser?Pro
1 5 10 15
Ile?Lys?Asp?Gln?Lys?Asn?Cys?Asn?Cys?Cys?Trp?Ala?Met?Ala?Ala?Ala
20 25 30
Gly?Asn?Ile?Glu?Thr?Leu?Trp?Arg?Ile?Ser?Phe?Trp?Asp?Phe?Val?Asp
35 40 45
Val?Ser?Val?His?Glu?Leu?Leu?Asp?Cys?Gly?Arg?Cys?Gly?Asp?Gly?Cys
50 55 60
His?Gly?Gly?Phe?Val?Trp?Asp?Ala?Phe?Ile?Thr?Val?Leu?Asn?Asn?Ser
65 70 75 80
Gly?Leu?Ala?Ser?Glu?Lys?Asp?Tyr?Pro?Phe?Gln?Gly?Lys?Val?Arg?Ala
85 90 95
His?Arg?Cys?His?Pro?Lys?Lys?Tyr?Gln?Lys?Val?Ala?Trp?Ile?Gln?Asp
100 105 110
Phe?Ile?Met?Leu?Gln?Asn?Asn?Glu?His?Arg?Ile?Ala?Gln?Tyr?Leu?Ala
115 120 125
Thr?Tyr?Gly?Pro?Ile?Thr?Val?Thr?Ile?Asn?Met?Lys?Pro?Leu?Gln?Leu
130 135 140
Tyr?Arg?Lys?Gly?Val?Ile?Lys?Ala?Thr?Pro?Thr?Thr?Cys?Asp?Pro?Gln
145 150 155 160
Leu?Val?Asp?His?Ser?Val?Leu?Leu?Val?Gly?Phe?Gly?Ser?Val?Lys?Ser
165 170 175
Glu?Glu?Gly?Ile?Trp?Ala?Glu?Thr?Val?Ser?Ser?Gln?Ser?Gln?Pro?Gln
180 185 190
Pro?Pro?His?Pro?Thr?Pro?Tyr?Trp?Ile?Leu?Lys?Asn?Ser?Trp?Gly?Ala
195 200 205
Gln?Trp?Gly?Glu?Lys?Gly?Tyr?Phe?Arg?Leu?His?Arg?Gly?Ser?Asn?Thr
210 215 220
Cys?Gly?Ile?Thr?Lys?Phe?Pro?Leu?Thr?Ala?Arg?Val?Gln?Lys?Pro?Asp
225 230 235 240
Met?Lys?Pro?Arg?Val?Ser?Cys?Pro?Pro
245
<210>9
<211>242
<212>PRT
＜213〉people
<400>9
Leu?Pro?Lys?Ser?Trp?Asp?Trp?Arg?Asn?Val?Asp?Gly?Val?Asn?Tyr?Ala
1 5 10 15
Ser?Ile?Thr?Arg?Asn?Gln?His?Ile?Pro?Gln?Tyr?Cys?Gly?Ser?Cys?Trp
20 25 30
Ala?His?Ala?Ser?Thr?Ser?Ala?Met?Ala?Asp?Arg?Ile?Asn?Ile?Lys?Arg
35 40 45
Lys?Gly?Ala?Trp?Pro?Ser?Thr?Leu?Leu?Ser?Val?Gln?Asn?Val?Ile?Asp
50 55 60
Cys?Gly?Asn?Ala?Gly?Ser?Cys?Glu?Gly?Gly?Asn?Asp?Leu?Ser?Val?Trp
65 70 75 80
Asp?Tyr?Ala?His?Gln?His?Gly?Ile?Pro?Asp?Glu?Thr?Cys?Asn?Asn?Tyr
85 90 95
Gln?Ala?Lys?Asp?Gln?Glu?Cys?Asp?Lys?Phe?Asn?Gln?Cys?Gly?Thr?Cys
100 105 110
Asn?Glu?Phe?Lys?Glu?Cys?His?Ala?Ile?Arg?Asn?Tyr?Thr?Leu?Trp?Arg
115 120 125
Val?Gly?Asp?Tyr?Gly?Ser?Leu?Ser?Gly?Arg?Glu?Lys?Met?Met?Ala?Glu
130 135 140
Ile?Tyr?Ala?Asn?Gly?Pro?Ile?Ser?Cys?Gly?Ile?Met?Ala?Thr?Glu?Arg
145 150 155 160
Leu?Ala?Asn?Tyr?Thr?Gly?Gly?Ile?Tyr?Ala?Glu?Tyr?Gln?Asp?Thr?Thr
165 170 175
Tyr?Ile?Asn?His?Val?Val?Ser?Val?Ala?Gly?Trp?Gly?Ile?Ser?Asp?Gly
180 185 190
Thr?Glu?Tyr?Trp?Ile?Val?Arg?Asn?Ser?Trp?Gly?Glu?Pro?Trp?Gly?Glu
195 200 205
Arg?Gly?Trp?Leu?Arg?Ile?Val?Thr?Ser?Thr?Tyr?Lys?Asp?Gly?Lys?Gly
210 215 220
Ala?Arg?Tyr?Asn?Leu?Ala?Ile?Glu?Glu?His?Cys?Thr?Phe?Gly?Asp?Pro
225 230 235 240
Ile?Val
<210>10
<211>221
<212>PRT
＜213〉people
<400>10
Leu?Pro?Lys?Ser?Val?Asp?Trp?Arg?Lys?Lys?Gly?Tyr?Val?Thr?Pro?Val
1 5 10 15
Lys?Asn?Gln?Lys?Gln?Cys?Gly?Ser?Cys?Trp?Ala?Phe?Ser?Ala?Thr?Gly
20 25 30
Ala?Leu?Glu?Gly?Gln?Met?Phe?Arg?Lys?Thr?Gly?Lys?Leu?Val?Ser?Leu
35 40 45
Ser?Glu?Gln?Asn?Leu?Val?Asp?Cys?Ser?Arg?Pro?Gln?Gly?Asn?Gln?Gly
50 55 60
Cys?Asn?Gly?Gly?Phe?Met?Ala?Arg?Ala?Phe?Gln?Tyr?Val?Lys?Glu?Asn
65 70 75 80
Gly?Gly?Leu?Asp?Ser?Glu?Glu?Ser?Tyr?Pro?Tyr?Val?Ala?Val?Asp?Glu
85 90 95
Ile?Cys?Lys?Tyr?Arg?Pro?Glu?Asn?Ser?Val?Ala?Asn?Asp?Thr?Gly?Phe
100 105 110
Thr?Val?Val?Ala?Pro?Gly?Lys?Glu?Lys?Ala?Leu?Met?Lys?Ala?Val?Ala
115 120 125
Thr?Val?Gly?Pro?Ile?Ser?Val?Ala?Met?Asp?Ala?Gly?His?Ser?Ser?Phe
130 135 140
Gln?Phe?Tyr?Lys?Ser?Gly?Ile?Tyr?Phe?Glu?Pro?Asp?Cys?Ser?Ser?Lys
145 150 155 160
Asn?Leu?Asp?His?Gly?Val?Leu?Val?Val?Gly?Tyr?Gly?Phe?Glu?Gly?Ala
165 170 175
Asn?Ser?Asn?Asn?Ser?Lys?Tyr?Trp?Leu?Val?Lys?ASh?Ser?Trp?Gly?Pro
180 185 190
Glu?Trp?Gly?Ser?Asn?Gly?Tyr?Val?Lys?Ile?Ala?Lys?Asp?Lys?Asn?Asn
195 200 205
His?Cys?Gly?Ile?Ala?Thr?Ala?Ala?Ser?Tyr?Pro?Asn?Val
210 215 220
<210>11
<211>237
<212>PRT
＜213〉little sickle plasmodium
<400>11
Val?Pro?Glu?Ile?Leu?Asp?Tyr?Arg?Glu?Lys?Gly?Ile?Val?His?Glu?Pro
1 5 10 15
Lys?Asp?Gln?Gly?Leu?Cys?Gly?Ser?Cys?Trp?Ala?Phe?Ala?Ser?Val?Gly
20 25 30
Asn?Ile?Glu?Ser?Val?Phe?Ala?Lys?Lys?Asn?Lys?Asn?Ile?Leu?Ser?Phe
35 40 45
Ser?Glu?Gln?Glu?Val?Val?Asp?Cys?Ser?Lys?Asp?Asn?Phe?Gly?Cys?Asp
50 55 60
Gly?Gly?His?Pro?Phe?Tyr?Ser?Phe?Leu?Tyr?Val?Leu?Gln?Asn?Glu?Leu
65 70 75 80
Cys?Leu?Gly?Asp?Glu?Tyr?Lys?Tyr?Lys?Ala?Lys?Asp?Asp?Met?Phe?Cys
85 90 95
Leu?Asn?Tyr?Arg?Cys?Lys?Arg?Lys?Val?Ser?Leu?Ser?Ser?Ile?Gly?Ala
100 105 110
Val?Lys?Glu?Asn?Gln?Leu?Ile?Leu?Ala?Leu?Asn?Glu?Val?Gly?Pro?Leu
115 120 125
Ser?Val?Asn?Val?Gly?Val?Ash?Asn?Asp?Phe?Val?Ala?Tyr?Ser?Glu?Gly
130 135 140
Val?Tyr?Asn?Gly?Thr?Cys?Ser?Glu?Glu?Leu?Asn?His?Ser?Val?Leu?Leu
145 150 155 160
Val?Gly?Tyr?Gly?Gln?Val?Glu?Lys?Thr?Lys?Leu?Asn?Tyr?Asn?Asn?Lys
165 170 175
Ile?Gln?Thr?Tyr?Asn?Thr?Lys?Glu?Asn?Ser?Asn?Gln?Pro?Asp?Asp?Asn
180 185 190
Ile?Ile?Tyr?Tyr?Trp?Ile?Ile?Lys?Asn?Ser?Trp?Ser?Lys?Lys?Trp?Gly
195 200 205
Glu?Asn?Gly?Phe?Met?Arg?Leu?Ser?Arg?Asn?Lys?Asn?Gly?Asp?Asn?Val
210 215 220
Phe?Cys?Gly?Ile?Gly?Glu?Glu?Val?Phe?Tyr?Pro?Ile?Leu
225 230 235
<210>12
<211>222
<212>PRT
＜213〉little sickle plasmodium
<400>12
Asp?Arg?Ile?Ala?Tyr?Asp?Trp?Arg?Leu?His?Gly?Gly?Val?Thr?Pro?Val
1 5 10 15
Lys?Asp?Gln?Ala?Leu?Cys?Gly?Ser?Cys?Trp?Ala?Phe?Ser?Ser?Val?Gly
20 25 30
Ser?Val?Glu?Ser?Gln?Tyr?Ala?Ile?Arg?Lys?Leu?Phe?Leu?Phe?Ser?Glu
35 40 45
Gln?Glu?Leu?Val?Asp?Cys?Ser?Val?Lys?Asn?Ash?Gly?Cys?Tyr?Gly?Gly
50 55 60
Tyr?Ile?Thr?Asn?Ala?Phe?Asp?Asp?Met?Ile?Asp?Leu?Gly?Gly?Leu?Cys
65 70 75 80
Ser?Gln?Asp?Asp?Tyr?Pro?Tyr?Val?Ser?Asn?Leu?Pro?Glu?Thr?Cys?Asn
85 90 95
Leu?Lys?Arg?Cys?Asn?Glu?Arg?Tyr?Thr?Ile?Lys?Ser?Tyr?Val?Ser?Ile
100 105 110
Pro?Asp?Asp?Lys?Phe?Lys?Glu?Ala?Leu?Arg?Tyr?Leu?Gly?Pro?Ile?Ser
115 120 125
Ile?Ser?Ile?Ala?Ala?Ser?Asp?Asp?Phe?Ala?Phe?Tyr?Arg?Gly?Gly?Phe
130 135 140
Tyr?Asp?Gly?Glu?Cys?Gly?Ala?Ala?Pro?Asn?His?Ala?Val?Ile?Leu?Val
145 150 155 160
Gly?Tyr?Gly?Met?Lys?Asp?Ile?Tyr?Asn?Glu?Asp?Thr?Gly?Arg?Met?Glu
165 170 175
Lys?Phe?Tyr?Tyr?Tyr?Ile?Ile?Lys?Asn?Ser?Trp?Gly?Ser?Asp?Trp?Gly
180 185 190
Glu?Gly?Gly?Tyr?Ile?Ash?Leu?Glu?Thr?Asp?Glu?Asn?Gly?Tyr?Lys?Lys
195 200 205
Thr?Cys?Ser?Ile?Gly?Thr?Glu?Ala?Tyr?Val?Pro?Leu?Leu?Glu
210 215 220
<210>13
<211>224
<212>PRT
＜213〉little sickle plasmodium
<400>13
Asp?His?Ala?Ala?Tyr?Asp?Trp?Arg?Leu?His?Ser?Gly?Val?Thr?Pro?Val
1 5 10 15
Lys?Asp?Gln?Lys?Asn?Cys?Gly?Ser?Cys?Trp?Ala?Phe?Ser?Ser?Ile?Gly
20 25 30
Ser?Val?Glu?Ser?Gln?Tyr?Ala?Ile?Arg?Lys?Asn?Lys?Leu?Ile?Thr?Leu
35 40 45
Ser?Glu?Gln?Glu?Leu?Val?Asp?Cys?Ser?Phe?Lys?Asn?Tyr?Gly?Cys?Asn
50 55 60
Gly?Gly?Leu?Ile?Asn?Ash?Ala?Phe?Glu?Asp?Met?Ile?Glu?Leu?Gly?Gly
65 70 75 80
Ile?Cys?Pro?Asp?Gly?Asp?Tyr?Pro?Tyr?Val?Ser?Asp?Ala?Pro?Asn?Leu
85 90 95
Cys?Asn?Ile?Asp?Arg?Cys?Thr?Glu?Lys?Tyr?Gly?Ile?Lys?Asn?Tyr?Leu
100 105 110
Ser?Val?Pro?Asp?Ash?Lys?Leu?Lys?Glu?Ala?Leu?Arg?Phe?Leu?Gly?Pro
115 120 125
Ile?Ser?Ile?Ser?Val?Ala?Val?Ser?Asp?Asp?Phe?Ala?Phe?Tyr?Lys?Glu
130 135 140
Gly?Ile?Phe?Asp?Gly?Glu?Cys?Gly?Asp?Glu?Leu?Ash?His?Ala?Val?Met
145 150 155 160
Leu?Val?Gly?Phe?Gly?Met?Lys?Glu?Ile?Val?Ash?Pro?Leu?Thr?Lys?Lys
165 170 175
Gly?Glu?Lys?His?Tyr?Tyr?Tyr?Ile?Ile?Lys?Asn?Ser?Trp?Gly?Gln?Gln
180 185 190
Trp?Gly?Glu?Arg?Gly?Phe?Ile?Asn?Ile?Glu?Thr?Asp?Glu?Ser?Gly?Leu
195 200 205
Met?Arg?Lys?Cys?Gly?Leu?Gly?Thr?Asp?Ala?Phe?Ile?Pro?Leu?Ile?Glu
210 215 220

Claims (24)

1, a kind of compound, it has following chemical formula:
Figure A2004800245200002C1
And have and be no more than 70 non-hydrogen atoms that are selected from C, O, N, S, P, F, Cl, Br or I independently of one another;
R wherein 4Be non-hydrogen electron-withdrawing substituent, like this so that itself and R 1, R 2And R 2Make the alkalescence of nitrogen be reduced to pKa together less than 6;
The wherein molecule of compound and tissue protein enzyme interacting, thus make in the chemical formula the CH-NH zone advantageously and the kethepsin between S2 and the S3 interact R 1Advantageously with the reactive site S1 of tissue protein zymophore but not reactive site S3 interacts R 2Advantageously with the S2 of kethepsin but not S3 interacts and R 2Advantageously with the S3 of tissue protein zymophore but not S2 or S1 interact.
2, the compound of claim 1, wherein kethepsin is selected from cathepsin B, F, H, K, L, L 2, O, S, W or Z.
3, the compound of claim 2, wherein kethepsin is selected from cathepsin K, L, S or B.
4, the compound of claim 1, wherein R 4Advantageously not respectively with the sublocus S of tissue protein zymophore 2, S 3And S 1Interact.
5, the compound of claim 1, wherein R 2Have at least one carbon atom that satisfies following three criterion distance simultaneously or sulphur atom, wherein said criterion distance is: it is at the C of kethepsin α 267 scopes in, at the C of kethepsin α 688.5 scopes in and at the C of kethepsin α 1347 scopes in.
6, the compound of claim 1, wherein R 3Have at least one carbon atom that satisfies following two criterion distance simultaneously or sulphur atom, wherein said criterion distance is: it is at the α of cathepsin C 665.5 scopes in and at the α of cathepsin C 607 scopes in.
7, the kethepsin amidocarbonylation that the compound of claim 1, wherein said nitrogen have less than the glycine 66 of 6 pKa value and described nitrogen and kethepsin forms a hydrogen bond.
8, the compound of claim 1, wherein R 2Comprise apolar regions.
9, the compound of claim 1, wherein R 2Comprise lipophilic zone.
10, the compound of claim 1, wherein R 3Comprise apolar regions.
11, the compound of claim 1, wherein R 3Comprise lipophilic zone.
12, the compound of claim 1, wherein the pKa of the nitrogen of the secondary amine shown in the claim 1 in water medium is less than 5.
13, the compound of claim 1, wherein R 4Be to be selected from-CF 3,-CH 2F ,-CF 2R 5With-CHFR 5Group, R wherein 5Be the C that is randomly replaced by 1～4 substituting group 1-6Alkyl, aryl or heteroaryl, described substituting group is selected from halogen, C 1-3Alkyl, C 1-3Alkoxyl group, hydroxyl, hydroxyalkyl, ketone, cyano group, heterocyclic radical, C 3-8Cycloalkyl, SO mC 1-3Alkyl, NH 2, NO 2Perhaps O (C=O) C 1-3Alkyl; With m be 0～2 integer.
14, the compound of claim 1, wherein R 1Comprise the zone that stably cooperates with the sublocus S1 of tissue protein zymophore, and at the α of cathepsin C 255 scopes in have at least 1 carbon atom.
15, the compound of claim 14, wherein kethepsin is selected from cathepsin B, F, H, K, L, L 2, O, S, W or Z.
16, the compound of claim 14, wherein the sulphur of the halfcystine 25 of compound and kethepsin forms covalent linkage.
17, the compound of claim 14, wherein R 1Right and wrong are immunogenic.
18, the compound of claim 14, wherein compound combines with the reactive site of kethepsin, has in the enzymatic determination of purifying less than 10 micromolar IC 50
19, the compound of claim 14, wherein the close electric carbonyl carbon with compound forms a covalent linkage.
20, the compound of claim 1 does not wherein form covalent linkage between compound and kethepsin.
21, pharmaceutical composition, comprise with pharmaceutically acceptable carrier combinations as each defined compound or its pharmacy acceptable salt in the claim 1 to 20.
22, the purposes in making medicine as each defined compound of claim 1 to 20 or its pharmacy acceptable salt, wherein said medicine is used for the inhibition of histone enzymic activity, perhaps treatment or the kethepsin dependent state of prevention in the Mammals, the bone that perhaps suppresses or reduce in the Mammals loses, perhaps treatment or the osteoporosis of prevention in the Mammals, perhaps treatment or the rheumatic arthritis state of prevention in the Mammals, perhaps treat or prevent the carrying out of osteoarthritis in the Mammals, perhaps treat the cancer in the Mammals.
23, be used for pharmacological agent as each defined compound or its pharmacy acceptable salt in the claim 1 to 20.
24, a kind ofly be used for the treatment of or prevent the kethepsin dependent status in the Mammals or suppress or reduce the carrying out of osteoarthritis in bone loss in the Mammals or the osteoporosis in treatment or the prevention Mammals or the rheumatic arthritis state in treatment or the prevention Mammals or treatment or the prevention Mammals, perhaps treat the method for cancer in the Mammals, it comprise to Mammals drug treatment significant quantity as each defined compound or its pharmacy acceptable salt in the claim 1 to 20.
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CN102014635A (en) * 2008-04-01 2011-04-13 维罗贝股份有限公司 Di-fluoro containing compounds as cysteine protease inhibitors
US8748649B2 (en) 2006-10-04 2014-06-10 Virobay, Inc. Di-fluoro containing compounds as cysteine protease inhibitors
TWI483720B (en) * 2006-10-04 2015-05-11 Virobay Inc Di-fluoro containing compounds as cysteine protease inhibitors
CN106866502A (en) * 2015-12-10 2017-06-20 广东东阳光药业有限公司 Cathepsin K inhibitor and application thereof
CN108464976A (en) * 2009-06-22 2018-08-31 扩散药品有限公司 Diffusion promotes compound and its independent or application together with Thrombolytic Drugs
WO2020063821A1 (en) * 2018-09-30 2020-04-02 北京和理咨询有限公司 Polypeptide conjugate of odn or derivative thereof, preparation method therefor and application thereof

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US8748649B2 (en) 2006-10-04 2014-06-10 Virobay, Inc. Di-fluoro containing compounds as cysteine protease inhibitors
TWI483720B (en) * 2006-10-04 2015-05-11 Virobay Inc Di-fluoro containing compounds as cysteine protease inhibitors
CN102014635A (en) * 2008-04-01 2011-04-13 维罗贝股份有限公司 Di-fluoro containing compounds as cysteine protease inhibitors
CN108464976A (en) * 2009-06-22 2018-08-31 扩散药品有限公司 Diffusion promotes compound and its independent or application together with Thrombolytic Drugs
CN106866502A (en) * 2015-12-10 2017-06-20 广东东阳光药业有限公司 Cathepsin K inhibitor and application thereof
CN106866502B (en) * 2015-12-10 2020-10-09 广东东阳光药业有限公司 Cathepsin K inhibitors and uses thereof
WO2020063821A1 (en) * 2018-09-30 2020-04-02 北京和理咨询有限公司 Polypeptide conjugate of odn or derivative thereof, preparation method therefor and application thereof

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